Mammalian galanin receptors

ABSTRACT

The present invention provides isolated mammalian GalR3 receptors, isolated or recombinant nucleic acids and recombinant vectors encoding the same, host cells comprising the nucleic acids and vectors, and methods for making the receptors using the host cells. This invention further provides antibodies and antigen binding fragments thereof which specifically bind to the receptors and are useful for treating medical conditions caused or mediated by galanin. Also provided are screening methods for identifying specific agonists and antagonists of the mammalian GalR3 receptors.

TECHNICAL FIELD

[0001] The present invention relates to mammalian galanin receptors.More particularly, it relates to rat and human galanin receptors,isolated nucleic acids and recombinant vectors encoding the receptors,methods for making the receptors, fragments or fusion proteins thereofusing recombinant DNA methodology or chemical synthesis, and to methodsfor using the receptors in screening systems to identify inhibitors forthe treatment of various diseases. This invention further relates toantibodies, both polyclonal and monoclonal, which specifically bind tothe galanin receptors or to anti-idiotypic antibodies against them, andto fragments and fusion proteins thereof.

BACKGROUND OF THE INVENTION

[0002] Galanin is a polypeptide found in the central and peripheralnervous systems which regulates multiple processes such as endocrine andexocrine pancreatic secretions, intestinal motility, and modulation ofbehavioral, cognitive, and sensory functions such as feeding, learning,memory and nociception. See, e.g., Merchenthaler et al., Prog.Neurobiol. 40:711-769 (1993), and Hökfelt et al. in Galanin: A NewMultifunctional Peptide in the Neuro-Endocrine System, Wenner-GrenInternational Symposium Series, 1991, Vol. 58, MacMillan, Cambridge,U.K. Because of its wide 25 distribution and multiple activities,Galanin is believed to be involved in a number of medical conditions,including obesity, Alzheimer's disease, nociception, dementia, eatingdisorders, diabetes, dislipoproteinemia, developmental disorders of theneural systems, disorders of the digestive systems, growth disorders,sexual and reproductive dysfunctions, stomach ulcers, sleep disorders,and regeneration of injured neuronal systems.

[0003] The physiological effects of galanin are mediated by specificreceptors in target tissues. One such receptor from insulin-secretingcells has been described by Lagny-Pourmir et al. [Endocrinology124:2635-2641 (1989)]. Human galanin receptors have been cloned byHabert-Ortoli et al. [Proc. Natl. Acad. Sci. USA 91:9780-9783 (1994)],Hinuma et al. [European Patent Application Publication EP 0 711 830 A2]and Amiranoff et al. [International Patent Application Publication No.WO 95/22608].

[0004] In view of the important role of galanin in many physiologicalprocesses and medical conditions, there is a need for materials andmethods for identifying selective agonists and antagonists of galanin.

SUMMARY OF THE INVENTION

[0005] The present invention fills the foregoing need by providing suchmaterials and methods. More particularly, this invention provides novelmammalian galanin receptors, isolated or recombinant nucleic acidsencoding the receptors, and recombinant vectors and host cellscomprising such nucleic acids.

[0006] The isolated or recombinant nucleic acids are selected from thegroup consisting of:

[0007] (a) a nucleic acid encoding a mammalian galanin receptorcomprising an amino acid sequence defined by SEQ ID NO: 2 or SEQ ID NO:4, or a subsequence thereof;

[0008] (b) a nucleic acid that hybridizes under moderately stringentconditions to the nucleic acid of (a) and encodes a polypeptide that (i)binds galanin and (ii) is at least 80% identical to a receptor encodedby the nucleic acid of (a); and

[0009] (c) a nucleic acid that, due to the degeneracy of the geneticcode, encodes a mammalian galanin receptor encoded by a nucleic acid of(a) or (b).

[0010] This invention further provides methods for making the galaninreceptors comprising culturing a host cell comprising a nucleic acidencoding a mammalian galanin receptor comprising an amino acid sequencedefined by SEQ ID NO: 2 or SEQ ID NO: 4, or a subsequence thereof, underconditions in which the nucleic acid is expressed. In some embodiments,the method further comprises isolation of the receptor from the culture.

[0011] This invention also provides polypeptides comprising a fragmentof a mammalian galanin receptor having an amino acid sequencecorresponding to the sequence of at least about 8 contiguous residues ofthe complete receptor sequence. Preferably, the polypeptides comprise atleast about 12, more preferably at least about 20, and most preferablyat least about 30 such residues.

[0012] Still further, this invention provides fusion proteins comprisinga mammalian galanin receptor or a polypeptide therefrom covalentlylinked to a fusion partner.

[0013] The present invention also provides antibodies, both polyclonaland monoclonal, that specifically bind to one or more of the galaninreceptors or to polypeptides therefrom, and anti-idiotypic antibodies,both monoclonal and polyclonal, which specifically bind to the foregoingantibodies.

[0014] This invention further provides a method for producing amammalian galanin receptor comprising culturing a host cell comprising anucleic acid encoding a mammalian galanin receptor comprising an aminoacid sequence defined by SEQ ID NO: 2 or SEQ ID NO: 4, or a subsequencethereof, under conditions in which the nucleic acid is expressed. In oneembodiment the receptor is isolated from the culture.

[0015] This invention still further provides a method for treatinggalanin-mediated medical conditions comprising administering to a mammalafflicted with a medical condition caused or mediated by galanin, aneffective amount of an antibody, or an antigen-binding fragment thereof,that specifically binds to a mammalian galanin receptor having an aminoacid sequence defined by SEQ ID NO: 4, or a subsequence thereof, andpharmaceutical compositions comprising one or more of such antibodies orfragments and a pharmaceutically acceptable carrier. Preferably, themammal is a human being.

[0016] The present invention also provides a method for identifying agalanin agonist or antagonist comprising:

[0017] (a) contacting a mammalian galanin receptor having an amino acidsequence defined by SEQ ID NO: 2 or SEQ ID NO: 4, or a subsequencethereof, in the presence of a known amount of labeled galanin with asample to be tested for the presence of a galanin agonist or antagonist;and

[0018] (b) measuring the amount of labeled galanin specifically bound tothe receptor;

[0019] whereby a galanin agonist or antagonist in the sample isidentified by measuring substantially reduced binding of the labeledgalanin to the galanin receptor, compared to what would be measured inthe absence of such agonist or antagonist.

[0020] In a preferred embodiment, membranes isolated from mammaliancells comprising a nucleic acid encoding the galanin receptor are usedas the source of the receptor.

BRIEF DESCRIPTION OF THE FIGURES

[0021] The present invention can be more readily understood by referenceto the following Description and Examples, and to the accompanyingFigures, in which:

[0022]FIG. 1 is a graphical representation of the specific binding of¹²⁵I-galanin to cellular receptors, showing bound radioactivity as afunction of ligand concentration; and

[0023]FIG. 2 is a graphical representation of the inhibition of thebinding of ¹²⁵I-porcine galanin to cellular receptors by various knowngalanin agonists and antagonists, showing percent inhibition ofradioligand binding as a function of ligand concentration. The competingligands were rat galanin (), chimeric galanin peptide C7 (◯),galanin(2-29) (□), galanin(3-29) (Δ) and galanin(10-20) (▴).

DESCRIPTION OF THE INVENTION

[0024] All references cited herein are hereby incorporated herein intheir entirety by reference.

[0025] As used herein, the term “ligand” is defined to mean any moleculecapable of specifically binding to the mammalian galanin receptors ofthe invention. Thus galanin itself is a ligand, as are agonists andantagonists that may compete with galanin for specific binding to thereceptors.

[0026] Galanin Receptor Characterization

[0027] As has been noted above, others had identified galanin receptorsfrom various species and tissues prior to the present invention. Thus,there appears to be a family of galanin receptor subtypes. The humanreceptor cloned by Habert-Ortoli et al., supra, was the first and hencehas been called the type 1 galanin receptor (GalR1). Homologous GalR1receptors have been cloned from rat Rin14B insulinoma cells [Parker etal., Mol. Brain Res. 34:179-189 (1995)] and from rat brain [Burgevin etal., J. Mol. Neurosci. 6:33-41 (1995)].

[0028] More recently a second galanin receptor subtype, type 2 (GalR2),has been described in rat by Howard et al. [FEBS Lett. 405:285-290(1997) and by Wang et al. [Mol. Pharmacol. Vol. 52:1 (1997) (in press)].Because of the previously known existence of type 1 and type 2 galaninreceptors, the novel galanin receptors of this invention may be referredto as type 3 galanin receptors (GalR3).

[0029] The nucleotide sequence of the complete open reading frame andthe corresponding amino acid sequence of rat GalR3 receptor cDNA aredefined in the Sequence Listing by SEQ ID NO: 1 and SEQ ID NO: 2,respectively. The nucleotide sequence of the complete open reading frameand the corresponding amino acid sequence of the human GalR3 receptorare defined in the Sequence Listing by SEQ ID NO: 3 and SEQ ID NO: 4,respectively.

[0030] In comparing the complete rat and human cDNA sequences, it hasbeen found that the human sequence is longer, and that a region encodedby bases 169 to 1275 (amino acid residues 61-424) of the complete humansequence is highly homologous to the rat sequence. The human GalR3receptor sequence may thus be regarded as having two forms—a “long” formencompassing the entire sequence defined by SEQ. ID NO: 4, and a “short”form encompassing the region encoded by bases 169 to 1275 of SEQ ID NO:3.

[0031] The long form occurs naturally, as the start and stop codons wereidentified from human brain cDNA that was reverse-transcribed fromnatural transcripts. The short form may result from the long form by amodifying mechanism, e.g., from an alternative splicing of thetranscript or from an independent transcript generated at a separategenomic locus, but whether that is true or not is irrelevant to thisinvention.

[0032] As used herein, the phrase an isolated or recombinant receptorcomprising an amino acid sequence “defined by SEQ ID NO: 4, or asubsequence thereof” is thus defined to include both “short” and “long”forms of the human GalR3 receptor.

[0033] The present invention also encompasses fragments, analogs andphysical variants of the receptors. As used herein, the term“polypeptide” or “peptide” means a fragment or segment, e.g., of amammalian galanin receptor having an amino acid sequence defined by SEQID NO: 2 or 4 which comprises a subsequence of the complete amino acidsequence of the receptor containing at least about 8, preferably atleast about 12, more preferably at least about 20, and most preferablyat least about 30 or more contiguous amino acid residues, up to andincluding the total number of residues in the complete receptor.

[0034] The polypeptides of the invention can comprise any part of thecomplete sequence of such a receptor. Thus, although they could beproduced by proteolytic cleavage of an intact receptor, they can also bemade by chemical synthesis or by the application of recombinant DNAtechnology and are not limited to polypeptides delineated by proteolyticcleavage sites. The polypeptides, either alone or cross-linked orconjugated to a carrier molecule to render them more immunogenic, areuseful as antigens to elicit the production of antibodies. Theantibodies can be used, e.g., in immunoassays of the intact receptors,for immunoaffinity purification, etc.

[0035] The term “analog(s)” means a mammalian galanin receptor of theinvention which has been modified by deletion, addition, modification orsubstitution of one or more amino acid residues in the wild-typereceptor. It encompasses allelic and polymorphic variants, and alsomuteins and fusion proteins which comprise all or a significant part ofsuch a mammalian galanin receptor, e.g., covalently linked via aside-chain group or terminal residue to a different protein, polypeptideor moiety (fusion partner).

[0036] Some amino acid substitutions are preferably “conservative”, withresidues replaced with physicochemically similar residues, such asGly/Ala, Asp/Glu, Val/Ile/Leu, Lys/Arg, Asn/Gln and Phe/Trp/Tyr. Analogshaving such conservative substitutions typically retain substantialgalanin binding activity. Other analogs, which have non-conservativesubstitutions such as Asn/Glu, Val/Tyr and His/Glu, may substantiallylack such activity. Nevertheless, such analogs are useful because theycan be used as antigens to elicit production of antibodies in animmunologically competent host. Because these analogs retain many of theepitopes (antigenic determinants) of the wild-type receptors from whichthey are derived, many antibodies produced against them can also bind tothe active-conformation or denatured wild-type receptors. Accordingly,such antibodies can also be used, e.g., for the immunopurification orimmunoassay of the wild-type receptors.

[0037] Some analogs are truncated variants in which residues have beensuccessively deleted from the amino- and/or carboxyl-termini, whilesubstantially retaining the characteristic ligand binding activity.

[0038] Modifications of amino acid residues may include but are notlimited to aliphatic esters or amides of the carboxyl terminus or ofresidues containing carboxyl side chains, O-acyl derivatives of hydroxylgroup-containing residues, and N-acyl derivatives of the amino-terminalamino acid or amino-group containing residues, e.g., lysine or arginine.

[0039] Other analogs are mammalian galanin receptors containingmodifications, such as incorporation of unnatural amino acid residues,or phosphorylated amino acid residues such as phosphotyrosine,phosphoserine or phosphothreonine residues. Other potentialmodifications include sulfonation, biotinylation, or the addition ofother moieties, particularly those which have molecular shapes similarto phosphate groups.

[0040] Analogs of the mammalian galanin receptors can be prepared bychemical synthesis or by using site-directed mutagenesis [Gillman etal., Gene 8:81 (1979); Roberts et al., Nature 328:731 (1987) or Innis(Ed.), 1990, PCR Protocols: A Guide to Methods and Applications,Academic Press, New York, N.Y.] or the polymerase chain reaction method[PCR; Saiki et al., Science 239:487 (1988)], as exemplified by Daughertyet al. [Nucleic Acids Res. 19:2471 (1991)] to modify nucleic acidsencoding the complete receptors. Adding epitope tags for purification ordetection of recombinant products is envisioned.

[0041] General techniques for nucleic acid manipulation and expressionthat can be used to make the analogs are described generally, e.g., inSambrook, et al., Molecular Cloning: A Laboratory Manual (2d ed.), 1989,Vols. 1-3, Cold Spring Harbor Laboratory. Techniques for the synthesisof polypeptides are described, for example, in Merrifield, J. Amer.Chem. Soc. 85:2149 (1963); Merrifield, Science 232:341 (1986); andAtherton et al., Solid Phase Peptide Synthesis: A Practical Approach,1989, IRL Press, Oxford.

[0042] Still other analogs are prepared by the use of agents known inthe art for their usefulness in cross-linking proteins through reactiveside groups. Preferred derivatization sites with cross-linking agentsare free amino groups, carbohydrate moieties and cysteine residues.

[0043] Substantial retention of ligand binding activity by the foregoinganalogs of the mammalian galanin receptors typically entails retentionof at least about 50%, preferably at least about 75%; more preferably atleast about 80%, and most preferably at least about 90% of the galaninbinding activity and/or specificity of the corresponding wild-typereceptor.

[0044] Some of the physical variants have substantial amino acidsequence homology with the amino acid sequences of the mammalian galaninreceptors or polypeptides. In this invention, amino acid sequencehomology, or sequence identity, is determined by optimizing residuematches and, if necessary, by introducing gaps as required. Homologousamino acid sequences are typically intended to include natural allelic,polymorphic and interspecies variations in each respective sequence.

[0045] Typical homologous proteins or peptides will have from 25-100%homology (if gaps can be introduced) to 50-100% homology (ifconservative substitutions are included), with the amino acid sequenceof the galanin receptors. Primate species receptors are of particularinterest.

[0046] Observed homologies will typically be at least about 35%,preferably at least about 50%, more preferably at least about 75%, andmost preferably at least about 80% or more. See Needleham et al., J.Mol. Biol. 48:443-453 (1970); Sankoff et al. in Time Warps, StringEdits, and Macromolecules: The Theory and Practice of SequenceComparison, 1983, Addison-Wesley, Reading, Mass.; and software packagesfrom IntelliGenetics, Mountain View, Calif., and the University ofWisconsin Genetics Computer Group, Madison, Wis.

[0047] Glycosylation variants include, e.g., analogs made by modifyingglycosylation patterns during synthesis and processing in variousalternative eukaryotic host expression systems, or during furtherprocessing steps. Particularly preferred methods for producingglycosylation modifications include exposing the mammalian galaninreceptors to glycosylating enzymes derived from cells which normallycarry out such processing, such as mammalian glycosylation enzymes.Alternatively, deglycosylation enzymes can be used to removecarbohydrates attached during production in eukaryotic expressionsystems.

[0048] Protein Purification

[0049] The proteins, polypeptides and antigenic fragments of thisinvention can be purified by standard methods, including but not limitedto salt or alcohol precipitation, preparative disc-gel electrophoresis,isoelectric focusing, high pressure liquid chromatography (HPLC),reversed-phase HPLC, gel filtration, cation and anion exchange andpartition chromatography, and countercurrent distribution. Suchpurification methods are well known in the art and are disclosed, e.g.,in Guide to Protein Purification, Methods in Enzymology, Vol. 182, M.Deutscher, Ed., 1990, Academic Press, New York, N.Y. More specificmethods applicable to purification of the galanin receptors aredescribed below.

[0050] Purification steps can be followed by carrying out assays forligand binding activity as described below. Particularly where areceptor is being isolated from a cellular or tissue source, it ispreferable to include one or more inhibitors of proteolytic enzymes isthe assay system, such as phenylmethanesulfonyl fluoride (PMSF).

[0051] Antibody Production

[0052] Antigenic (i.e., immunogenic) fragments of the mammalian galaninreceptors of this invention, which may or may not have ligand bindingactivity, may similarly be produced. Regardless of whether they bindgalanin, such fragments, like the complete receptors, are useful asantigens for preparing antibodies by standard methods that can bind tothe complete receptors. Shorter fragments can be concatenated orattached to a carrier. Because it is well known in the art that epitopesgenerally contain at least about five, preferably at least about 8,amino acid residues [Ohno et al., Proc. Natl. Acad. Sci. USA 82:2945(1985)], fragments used for the production of antibodies will generallybe at least that size. Preferably, they will contain even more residues,as described above. Whether a given fragment is immunogenic can readilybe determined by routine experimentation.

[0053] Although it is generally not necessary when complete mammaliangalanin receptors are used as antigens to elicit antibody production inan immunologically competent host, smaller antigenic fragments arepreferably first rendered more immunogenic by cross-linking orconcatenation, or by coupling to an immunogenic carrier molecule (i.e.,a macromolecule having the property of independently eliciting animmunological response in a host animal). Cross-linking or conjugationto a carrier molecule may be required because small polypeptidefragments sometimes act as haptens (molecules which are capable ofspecifically binding to an antibody but incapable of eliciting antibodyproduction, i.e., they are not immunogenic). Conjugation of suchfragments to an immunogenic carrier molecule renders them moreimmunogenic through what is commonly known as the “carrier effect”.

[0054] Suitable carrier molecules include, e.g., proteins and natural orsynthetic polymeric compounds such as polypeptides, polysaccharides,lipopolysaccharides etc. Protein carrier molecules are especiallypreferred, including but not limited to keyhole limpet hemocyanin andmammalian serum proteins such as human or bovine gammaglobulin, human,bovine or rabbit serum albumin, or methylated or other derivatives ofsuch proteins. Other protein carriers will be apparent to those skilledin the art. Preferably, but not necessarily, the protein carrier will beforeign to the host animal in which antibodies against the fragments areto be elicited.

[0055] Covalent coupling to the carrier molecule can be achieved usingmethods well known in the art, the exact choice of which will bedictated by the nature of the carrier molecule used. When theimmunogenic carrier molecule is a protein, the fragments of theinvention can be coupled, e.g., using water soluble carbodiimides suchas dicyclohexylcarbodiimide or glutaraldehyde.

[0056] Coupling agents such as these can also be used to cross-link thefragments to themselves without the use of a separate carrier molecule.Such cross-linking into aggregates can also increase. immunogenicity.Immunogenicity can also be increased by the use of known adjuvants,alone or in combination with coupling or aggregation.

[0057] Suitable adjuvants for the vaccination of animals include but arenot limited to Adjuvant 65 (containing peanut oil, mannide monooleateand aluminum monostearate); Freund's complete or incomplete adjuvant;mineral gels such as aluminum hydroxide, aluminum phosphate and alum;surfactants such as hexadecylamine, octadecylamine, lysolecithin,dimethyldioctadecylammonium bromide,N,N-dioctadecyl-N′,N′-bis(2-hydroxymethyl) propanediamine,methoxyhexadecylglycerol and pluronic polyols; polyanions such as pyran,dextran sulfate, poly IC, polyacrylic acid and carbopol; peptides suchas muramyl dipeptide, dimethylglycine and tuftsin; and oil emulsions.The polypeptides could also be administered following incorporation intoliposomes or other microcarriers.

[0058] Information concerning adjuvants and various aspects ofimmunoassays are disclosed, e.g., in the series by P. Tijssen, Practiceand Theory of Enzyme Immunoassays, 3rd Edition, 1987, Elsevier, N.Y.Other useful references covering methods for preparing polyclonalantisera include Microbiology, 1969, Hoeber Medical Division, Harper andRow; Landsteiner, Specificity of Serological Reactions, 1962, DoverPublications, New York, and Williams, et al., Methods in Immunology andImmunochemistry, Vol. 1, 1967, Academic Press, New York.

[0059] Serum produced from animals immunized using standard methods canbe used directly, or the IgG fraction can be separated from the serumusing standard methods such as plasmaphoresis or adsorptionchromatography with IgG-specific adsorbents such as immobilized ProteinA. Alternatively, monoclonal antibodies can be prepared.

[0060] Hybridomas producing monoclonal antibodies against the galaninreceptors of the invention or antigenic fragments thereof are producedby well-known techniques. Usually, the process involves the fusion of animmortalizing cell line with a B-lymphocyte that produces the desiredantibody. Alternatively, non-fusion techniques for generating immortalantibody-producing cell lines can be used, e.g., virally-inducedtransformation [Casali et al., Science 234:476 (1986)]. Immortalizingcell lines are usually transformed mammalian cells, particularly myelomacells of rodent, bovine, and human origin. Most frequently, rat or mousemyeloma cell lines are employed as a matter of convenience andavailability.

[0061] Techniques for obtaining antibody-producing lymphocytes frommammals injected with antigens are well known. Generally, peripheralblood lymphocytes (PBLs) are used if cells of human origin are employed,or spleen or lymph node cells are used from non-human mammalian sources.A host animal is injected with repeated dosages of the purified antigen(human cells are sensitized in vitro), and the animal is permitted togenerate the desired antibody-producing cells before they are harvestedfor fusion with the immortalizing cell line. Techniques for fusion arealso well known in the art, and in general involve mixing the cells witha fusing agent, such as polyethylene glycol.

[0062] Hybridomas are selected by standard procedures, such as HAT(hypoxanthine-aminopterin-thymidine) selection. Those secreting thedesired antibody are selected using standard immunoassays, such asWestern blotting, ELISA (enzyme-linked immunosorbent assay), RIA(radioimmunoassay), or the like. Antibodies are recovered from themedium using standard protein purification techniques [Tijssen, Practiceand Theory of Enzyme Immunoassays (Elsevier, Amsterdam, 1985)].

[0063] Many references are available to provide guidance in applying theabove techniques [Kohler et al., Hybridoma Techniques (Cold SpringHarbor Laboratory, New York, 1980); Tijssen, Practice and Theory ofEnzyme Immunoassays (Elsevier, Amsterdam, 1985); Campbell, MonoclonalAntibody Technology (Elsevier, Amsterdam, 1984); Hurrell, MonoclonalHybridoma Antibodies: Techniques and Applications (CRC Press, BocaRaton, Fla., 1982)]. Monoclonal antibodies can also be produced usingwell known phage library systems. See, e.g., Huse, et al., Science246:1275 (1989); Ward, et al., Nature 341:544 (1989).

[0064] Antibodies thus produced, whether polyclonal or monoclonal, canbe used, e.g., in an immobilized form bound to a solid support by wellknown methods, to purify the receptors by immunoaffinity chromatography.

[0065] Antibodies against the antigenic fragments can also be used,unlabeled or labeled by standard methods, as the basis for immunoassaysof the mammalian galanin receptors. The particular label used willdepend upon the type of immunoassay. Examples of labels that can be usedinclude but are not limited to radiolabels such as ³²P, ¹²⁵I, ³H and¹⁴C; fluorescent labels such as fluorescein and its derivatives,rhodamine and its derivatives, dansyl and umbelliferone;chemiluminescers such as luciferia and 2,3-dihydrophthalazinediones; andenzymes such as horseradish peroxidase, alkaline phosphatase, lysozymeand glucose-6-phosphate dehydrogenase.

[0066] The antibodies can be tagged with such labels by known methods.For example, coupling agents such as aldehydes, carbodiimides,dimaleimide, imidates, succinimides, bisdiazotized benzadine and thelike may be used to tag the antibodies with fluorescent,chemiluminescent or enzyme labels. The general methods involved are wellknown in the art and are described, e.g., in Immunoassay: A PracticalGuide, 1987, Chan (Ed.), Academic Press, Inc., Orlando, Fla. Suchimmunoassays could be carried out, for example, on fractions obtainedduring purification of the receptors.

[0067] The antibodies of the present invention can also be used toidentify particular cDNA clones expressing the galanin receptors inexpression cloning systems.

[0068] Neutralizing antibodies specific for the ligand binding site of areceptor can also be used as antagonists (inhibitors) to block galaninbinding. Such neutralizing antibodies can readily be identified throughroutine experimentation, e.g., by using the radioligand binding assaydescribed infra. Antagonism of galanin activity can be accomplishedusing complete antibody molecules, or well known antigen bindingfragments such as Fab, Fc, F(ab)₂, and Fv fragments.

[0069] Definitions of such fragments can be found, e.g., in Klein,Immunology (John Wiley, New York, 1982); Parham, Chapter 14, in Weir,ed. Immunochemistry, 4th Ed. (Blackwell Scientific Publishers, Oxford,1986). The use and generation of antibody fragments has also beendescribed, e.g.: Fab fragments [Tijssen, Practice and Theory of EnzymeImmunoassays (Elsevier, Amsterdam, 1985)], Fv fragments [Hochman et al.,Biochemistry 12:1130 (1973); Sharon et al., Biochemistry 15:1591 (1976);Ehrlich et al., U.S. Pat. No. 4,355,023] and antibody half molecules(Auditore-Hargreaves, U.S. Pat. No. 4,470,925). Methods for makingrecombinant Fv fragments based on known antibody heavy and light chainvariable region sequences have further been described, e.g., by Moore etal. (U.S. Pat. No. 4,642,334) and by Pluckthun [Bio/Technology 9:545(1991)]. Alternatively, they can be chemically synthesized by standardmethods.

[0070] Anti-idiotypic antibodies, both polyclonal and monoclonal, canalso be produced using the antibodies elicited against the receptors asantigens. Such antibodies can be useful as they may mimic the receptors.

[0071] Nucleic Acids and Expression Vectors

[0072] As used herein, the term “isolated nucleic acid” means a nucleicacid such as an RNA or DNA molecule, or a mixed polymer, which issubstantially separated from other components that are normally found incells or in recombinant DNA expression systems. These components includebut are not limited to ribosomes, polymerases, serum components, andflanking genomic sequences. The term thus embraces a nucleic acid whichhas been removed from its naturally occurring environment, and includesrecombinant or cloned DNA isolates and chemically synthesized analogs oranalogs biologically synthesized by heterologous systems. Asubstantially pure molecule includes isolated forms of the molecule.

[0073] An isolated nucleic acid will generally be a homogeneouscomposition of molecules but may, in some embodiments, contain minorheterogeneity. Such heterogeneity is typically found at the ends ofnucleic acid coding sequences or in regions not critical to a desiredbiological function or activity.

[0074] A “recombinant nucleic acid” is defined either by its method ofproduction or structure. Some recombinant nucleic acids are thus made bythe use of recombinant DNA techniques which involve human intervention,either in manipulation or selection. Others are made by fusing twofragments not naturally contiguous to each other. Engineered vectors areencompassed, as well as nucleic acids comprising sequences derived usingany synthetic oligonucleotide process.

[0075] For example, a wild-type codon may be replaced with a redundantcodon encoding the same amino acid residue or a conservativesubstitution, while at the same time introducing or removing a nucleicacid sequence recognition site. Similarly, nucleic acid segmentsencoding desired functions may be fused to generate a single geneticentity encoding a desired combination of functions not found together innature. Although restriction enzyme recognition sites are often thetarget of such artificial manipulations, other site-specific targets,e.g., promoters, DNA replication sites, regulation sequences, controlsequences, or other useful features may be incorporated by design.Sequences encoding epitope tags for detection or purification asdescribed above may also be incorporated.

[0076] A nucleic acid “fragment” is defined herein as a nucleotidesequence comprising at least about 17, generally at least about 25,preferably at least about 35, more preferably at least about 45, andmost preferably at least about 55 or more contiguous nucleotides.

[0077] This invention further encompasses recombinant DNA molecules andfragments having sequences that are identical or highly homologous tothose described herein. The nucleic acids of the invention may beoperably linked to DNA segments which control transcription,translation, and DNA replication.

[0078] “Homologous nucleic acid sequences” are those which when alignedand compared exhibit significant similarities. Standards for homology innucleic acids are either measures for homology generally used in the artby sequence comparison or based upon hybridization conditions, which aredescribed in greater detail below.

[0079] Substantial nucleotide sequence homology is observed when thereis identity in nucleotide residues in two sequences (or in theircomplementary strands) when optimally aligned to account for nucleotideinsertions or deletions, in at least about 50%, preferably in at leastabout 75%, more preferably in at least about 90%, and most preferably inat least about 95% of the aligned nucleotides.

[0080] Substantial homology also exists when one sequence will hybridizeunder selective hybridization conditions to another. Typically,selective hybridization will occur when there is at least about 55%homology over a stretch of at least about 30 nucleotides, preferably atleast about 65% over a stretch 6f at least about 25 nucleotides, morepreferably at least about 75%, and most preferably at least about 90%over about 20 nucleotides. See, e.g., Kanehisa, Nucleic Acids Res.12:203 (1984).

[0081] The lengths of such homology comparisons may encompass longerstretches and in certain embodiments may cover a sequence of at leastabout 17, preferably at least about 25, more preferably at least about50, and most preferably at least about 75 nucleotide residues.

[0082] Stringency of conditions employed in hybridizations to establishhomology are dependent upon factors such as salt concentration,temperature, the presence of organic solvents, and other parameters.Stringent temperature conditions usually include temperatures in excessof about 30° C., often in excess of about 37° C., typically in excess ofabout 45° C., preferably in excess of about 55° C., more preferably inexcess of about 65° C., and most preferably in excess of about 70° C.Stringent salt conditions will ordinarily be less than about 1000 mM,usually less than about 500 mM, more usually less than about 400 mM,preferably less than about 300 mM, more preferably less than about 200mM, and most preferably less than about 150 mM. For example, saltconcentrations of 100, 50 and 20 mM are used. The combination of theforegoing parameters, however, is more important than the measure of anysingle parameter. See, e.g., Wetmur et al., J. Mol. Biol. 31:349 (1968).

[0083] The term “substantially pure” is defined herein to mean amammalian galanin receptor, nucleic acid or other material that is freefrom other contaminating proteins, nucleic acids, and other biologicalsderived from an original source organism or recombinant DNA expressionsystem. Purity may be assayed by standard methods and will typicallyexceed at least about 50%, preferably at least about 75%, morepreferably at least about 90%, and most preferably at least about 95%purity. Purity evaluation may be made on a mass or molar basis.

[0084] Nucleic acids encoding the galanin receptors or fragments thereofcan be prepared by standard methods. For example, DNA can be chemicallysynthesized using, e.g., the phosphoramidite solid support method ofMatteucci et al. [J. Am. Chem. Soc. 103:3185 (1981)], the method of Yooet al. [J. Biol. Chem. 764:17078 (1989)], or other well known methods.This can be done by sequentially linking a series of oligonucleotidecassettes comprising pairs of synthetic oligonucleotides, as describedbelow.

[0085] Of course, due to the degeneracy of the genetic code, manydifferent nucleotide sequences can encode the galanin receptors. Thecodons can be selected for optimal expression in prokaryotic oreukaryotic systems. Such degenerate variants are of course alsoencompassed by this invention.

[0086] Moreover, nucleic acids encoding the galanin receptors canreadily be modified by nucleotide substitutions, nucleotide deletions,nucleotide insertions, and inversions of nucleotide stretches. Suchmodifications result in novel DNA sequences which encode antigens havingimmunogenic or antigenic activity in common with the wild-typereceptors. These modified sequences can be used to produce wild-type ormutant receptors, or to enhance expression in a recombinant DNA system.

[0087] Insertion of the DNAs encoding the galanin receptors into avector is easily accomplished when the termini of both the DNAs and thevector comprise compatible restriction sites. If this cannot be done, itmay be necessary to modify the termini of the DNAs and/or vector bydigesting back single-stranded DNA overhangs generated by restrictionendonuclease cleavage to produce blunt ends, or to achieve the sameresult by filling in the single-stranded termini with an appropriate DNApolymerase.

[0088] Alternatively, desired sites may be produced, e.g., by ligatingnucleotide sequences (linkers) onto the termini. Such linkers maycomprise specific oligonucleotide sequences that define desiredrestriction sites. Restriction sites can also be generated by the use ofthe polymerase chain reaction (PCR). See, e.g., Saiki et al., Science239:487 (1988). The cleaved vector and the DNA fragments may also bemodified if required by homopolymeric tailing.

[0089] Recombinant expression vectors used in this invention aretypically self-replicating DNA or RNA constructs comprising nucleicacids encoding one of the mammalian GalR3 receptors, usually operablylinked to suitable genetic control elements that are capable ofregulating expression of the nucleic acids in compatible host cells.Genetic control elements may include a prokaryotic promoter system or aeukaryotic promoter expression control system, and typically include atranscriptional promoter, an optional operator to control the onset oftranscription, transcription enhancers to elevate the level of mRNAexpression, a sequence that encodes a suitable ribosome binding site,and sequences that terminate transcription and translation. Expressionvectors also may contain an origin of replication that allows the vectorto replicate independently of the host cell.

[0090] Vectors that could be used in this invention include microbialplasmids, viruses, bacteriophage, integratable DNA fragments, and othervehicles which may facilitate integration of the nucleic acids into thegenome of the host. Plasmids are the most commonly used form of vectorbut all other forms of vectors which serve an equivalent function andwhich are, or become, known in the art are suitable for use herein. See,e.g., Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985 andSupplements, Elsevier, N.Y., and Rodriguez et al. (eds.), Vectors: ASurvey of Molecular Cloning Vectors and Their Uses, 1988, Buttersworth,Boston, Mass.

[0091] Expression of nucleic acids encoding the galanin receptors ofthis invention can be carried out by conventional methods in eitherprokaryotic or eukaryotic cells. Although strains of E. coli areemployed most frequently in prokaryotic systems, many other bacteriasuch as various strains of Pseudomonas and Bacillus are know in the artand can be used as well.

[0092] Prokaryotic expression control sequences typically used includepromoters, including those derived from the β-lactamase and lactosepromoter systems [Chang et al., Nature 198:1056 (1977)], the tryptophan(trp) promoter system [Goeddel et al., Nucleic Acids Res. 8:4057(1980)], the lambda P_(L) promoter system [Shimatake et al., Nature292:128 (1981)] and the tac promoter [De Boer et al., Proc. Natl. Acad.Sci. USA 292:128 (1983)]. Numerous expression vectors containing suchcontrol sequences are known in the art and available commercially.

[0093] Suitable host cells for expressing nucleic acids encoding themammalian GalR3 receptors include prokaryotes and higher eukaryotes.Prokaryotes include both gram negative and positive organisms, e.g., E.coli and B. subtilis. Higher eukaryotes include established tissueculture cell lines from animal cells, both of non-mammalian origin,e.g., insect cells, and birds, and of mammalian origin, e.g., human,primates, and rodents.

[0094] Prokaryotic host-vector systems include a wide variety of vectorsfor many different species. As used herein, E. coli and its vectors willbe used generically to include equivalent vectors used in otherprokaryotes. A representative vector for amplifying DNA is pBR322 ormany of its derivatives. Vectors that can be used to express themammalian GalR3 receptors include but are not limited to thosecontaining the lac promoter (pUC-series); trp promoter (pBR322-trp); Ipppromoter (the pIN-series); lambda-pP or pR promoters (pOTS); or hybridpromoters such as ptac (pDR540). See Brosius et al., “Expression VectorsEmploying Lambda-, trp-, lac-, and Ipp-derived Promoters”, in Rodriguezand Denhardt (eds.) Vectors: A Survey of Molecular Cloning Vectors andTheir Uses, 1988, Buttersworth, Boston, pp. 205-236.

[0095] Higher eukaryotic tissue culture cells are preferred hosts forthe recombinant production of the mammalian GalR3 receptors.

[0096] Although any higher eukaryotic tissue culture cell line might beused, including insect baculovirus expression systems, mammalian cellsare preferred. Transformation or transfection and propagation of suchcells has become a routine procedure. Examples of useful cell linesinclude HeLa cells, Chinese hamster ovary (CHO) cell lines, baby ratkidney (BRK) cell lines, insect cell lines, bird cell lines, and monkey(COS) cell lines.

[0097] Expression vectors for such cell lines usually include an originof replication, a promoter, a translation initiation site, RNA splicesites (if genomic DNA is used), a polyadenylation site, and atranscription termination site. These vectors also usually contain aselection gene or amplification gene. Suitable expression vectors may beplasmids, viruses, or retroviruses carrying promoters derived, e.g.,from such sources as adenovirus, SV40, parvoviruses, vaccinia virus, orcytomegalovirus. Representative examples of suitable expression vectorsinclude pCR®3.1, pCDNA1, pCD [Okayama et al., Mol. Cell Biol. 5:1136(1985)], pMC1neo Poly-A [Thomas et al., Cell 51:503 (1987)], pUC19,pREP8, pSVSPORT and derivatives thereof, and baculovirus vectors such aspAC 373 or pAC 610.

[0098] Pharmaceutical Compositions

[0099] The antibodies and antigen-binding fragments thereof can be usedtherapeutically to block the activity of galanin, and thereby to treatany medical condition caused or mediated by galanin. Such antibodies andfragments are preferably chimeric or humanized, to reduce antigenicityand human anti-mouse antibody (HAMA) reactions. The methodology involvedis disclosed, e.g., in U.S. Pat. No. 4,816,397 to Boss et al. and inU.S. Pat. No. 4,816,567 to Cabilly et al. Further refinements onantibody humanization are described in European Patent 451 216 B1.

[0100] The dosage regimen involved in a therapeutic application will bedetermined by the attending physician, considering various factors whichmay modify the action of the antibodies or binding fragments, e.g., thecondition, body weight, sex and diet of the patient, the severity of anyinfection, time of administration, and other clinical factors.

[0101] Typical protocols for the therapeutic administration ofantibodies are well known in the art and have been disclosed, e.g., byElliott et al. [The Lancet 344:1125 (1994)], Isaacs et al. [The Lancet340:748 (1992)], Anasetti et al. [Transplantation 54:844 (1992)],Anasetti et al. [Blood 84:1320 (1994)], Hale et al. [The Lancet 2:1394(Dec. 17, 1988)], Queen [Scrip 1881:18 (1993)] and Mathieson et al. [N.Eng. J. Med. 323:250 (1990)].

[0102] Administration of the compositions of this invention is typicallyparenteral, by intraperitoneal, intravenous, subcutaneous, orintramuscular injection, or by infusion or by any other acceptablesystemic method. Administration by intravenous infusion, typically overa time course of about 1 to 5 hours, is preferred.

[0103] Often, treatment dosages are titrated upward from a low level tooptimize safety and efficacy. Generally, daily antibody dosages willfall within a range of about 0.01 to 20 mg protein per kilogram of bodyweight. Typically, the dosage range will be from about 0.1 to 5 mgprotein per kilogram of body weight.

[0104] Dosages of antigen binding fragments from the antibodies will beadjusted to account for the smaller molecular sizes and possiblydecreased half-lives (clearance times) following administration. Variousmodifications or derivatives of the antibodies or fragments, such asaddition of polyethylene glycol chains (PEGylation), may be made toinfluence their pharmacokinetic and/or pharmacodynamic properties.

[0105] It will be appreciated by those skilled in the art, however, thatthe galanin antagonists of the invention are not limited to neutralizingantibodies or binding fragments thereof. This invention also encompassesother types of inhibitors, including small organic molecules andinhibitory ligand analogs, which can be identified using the methods ofthe invention.

[0106] An “effective amount” of a composition of the invention is anamount that will ameliorate one or more of the well known parametersthat characterize medical conditions caused or mediated by galanin. Manysuch parameters and conditions have been described, e.g., as in reviewsby Bantfa (Psychopharmacology: The Fourth Generation of Progress, 1995,F. E. Bloom and D. J. Kupfer, Eds., Ravin Press, Ltd., New York, N.Y.,pp. 563-571) and Crawley [Life Science 58:2185-2199 (1996)].

[0107] Although the compositions of this invention could be administeredin simple solution, they are more typically used in combination withother materials such as carriers, preferably pharmaceutical carriers.Useful pharmaceutical carriers can be any compatible, non-toxicsubstance suitable for delivering the compositions of the invention to apatient. Sterile water, alcohol, fats, waxes, and inert solids may beincluded in a carrier. Pharmaceutically acceptable adjuvants (bufferingagents, dispersing agents) may also be incorporated into thepharmaceutical composition. Generally, compositions useful forparenteral administration of such drugs are well known; e.g. Remington'sPharmaceutical Science, 17th Ed. (Mack Publishing Company, Easton, Pa.,1990). Alternatively, compositions of the invention may be introducedinto a patient's body by implantable drug delivery systems [Urquhart etal., Ann. Rev. Pharmacol. Toxicol. 24:199 (1984)].

[0108] Therapeutic formulations may be administered in many conventionaldosage formulation. Formulations typically comprise at least one activeingredient, together with one or more pharmaceutically acceptablecarriers. Formulations may include those suitable for oral, rectal,nasal, or parenteral (including subcutaneous, intramuscular, intravenousand intradermal) administration.

[0109] The formulations may conveniently be presented in unit dosageform and may be prepared by any methods well known in the art ofpharmacy. See, e.g., Gilman et al. (eds.) (1990), The PharmacologicalBases of Therapeutics, 8th Ed., Pergamon Press; and Remington'sPharmaceutical Sciences, supra, Easton, Pa.; Avis et al. (eds.) (1993)Pharmaceutical Dosage Forms: Parenteral Medications Dekker, New York;Lieberman et al. (eds.) (1990) Pharmaceutical Dosage Forms: TabletsDekker, New York; and Lieberman et al. (eds.) (1990), PharmaceuticalDosage Forms: Disperse Systems Dekker, New York.

[0110] The present invention also encompasses anti-idiotypic antibodies,both polyclonal and monoclonal, which are produced using theabove-described antibodies as antigens. These antibodies are usefulbecause they may mimic the structures of the receptors.

[0111] Screening Systems and Methods

[0112] The galanin receptors of this invention can be employed inscreening systems to identify agonists or antagonists of the receptors.Essentially, these systems provide methods for bringing together amammalian galanin receptor, an appropriate known ligand, includinggalanin itself, and a sample to be tested for the presence of a galaninagonist or antagonist.

[0113] Two basic types of screening systems can be used, alabeled-ligand binding assay and a “functional” assay. A labeled ligandfor use in the binding assay can be obtained by labeling galanin or aknown galanin agonist with a measurable group as described above inconnection with the labeling of antibodies. Various labeled forms ofgalanin are available commercially. In an example below, ¹²⁵I-galanin isused as the ligand.

[0114] Typically, a given amount of one of the galanin receptors of theinvention is contacted with increasing amounts of a labeled ligand, suchas labeled galanin itself, and the amount of the bound labeled ligand ismeasured after removing unbound labeled ligand by washing. As the amountof the labeled ligand is increased, a point is eventually reached atwhich all receptor binding sites are occupied or saturated. A plot ofsuch binding is shown in FIG. 1. Specific receptor binding of thelabeled ligand is abolished by a large excess of unlabled ligand.

[0115] Preferably, an assay system is used in which non-specific bindingof the labeled ligand to the receptor is minimal. Non-specific bindingis typically less than 50%, preferably less than 15%, and morepreferably less than 10% of the total binding of the labeled ligand.

[0116] As used herein, the term “galanin ligand” is defined to meangalanin itself or a fragment thereof comprising at least about thefifteen amino-terminal residues of galanin, and extending up to thecomplete galanin molecule. The amino acid sequence of the amino-terminalresidues is conserved in the galanins of various species, includinghumans. Therefore, galanin from one species may bind to galaninreceptors from another species; e.g., porcine galanin binds to the ratreceptor, as is illustrated in an Example below. For regulatorypurposes, however, it may be desirable to use human galanin or an activefragment thereof as the galanin ligand in conjunction with the humanreceptor when screening for galanin agonists or antagonists for humantherapeutic purposes.

[0117] In principle, a binding assay of the invention could be carriedout using a soluble receptor of the invention, e.g., followingproduction and refolding by standard methods from an E. coli expressionsystem, and the resulting receptor-labeled ligand complex could beprecipitated, e.g., using an antibody against the receptor. Theprecipitate could then be washed and the amount of the bound labeledligand could be measured.

[0118] Preferably, however, a nucleic acid encoding one of the galaninreceptors of the invention is transfected into an appropriate host cell,whereby the receptor will become incorporated into the membrane of thecell. A membrane fraction can then be isolated from the cell and used asa source of the receptor for assay. Preferably, specific binding of thelabeled ligand to a membrane fraction from the untransfected host cellwill be negligible, as is the case with COS-7 cells used in an Examplebelow.

[0119] The binding assays of this invention can be used to identify bothgalanin agonists and antagonists, because both will compete for bindingto the receptor with the labeled ligand.

[0120] In the basic binding assay, the method for identifying a galaninagonist or antagonist comprises:

[0121] (a) contacting a mammalian GalR3 receptor having an amino acidsequence defined by SEQ ID NO: 2 or SEQ ID NO: 4, or a subsequencethereof, in the presence of a known amount of labled galanin with asample to be tested for the presence of a galanin agonist or antagonist;and

[0122] (b) measuring the amount of labeled galanin bound to thereceptor;

[0123] whereby a galanin agonist or antagonist in the sample isidentified by measuring substantially reduced binding of the labeledgalanin to the GalR3 receptor, compared to what would be measured in theabsence of such agonist or antagonist.

[0124] Preferably, the GalR3 receptor used to identify a galanin agonistor antagonist for human therapeutic purposes has an amino acid sequencedefined by SEQ ID NO: 4, or a subsequence thereof.

[0125] In one embodiment of the invention, the foregoing method furthercomprises:

[0126] (c) contacting a mammalian GalR1 or GalR2 receptor in thepresence of a known amount of labeled galanin with a compound identifiedas a galanin agonist or antagonist in steps (a) and (b); and

[0127] (d) measuring the amount of labeled galanin bound to thereceptor;

[0128] whereby a galanin agonist or antagonist specific for the GalR3receptor is identified by measuring substantially undiminished bindingof the labeled galanin to the receptor, compared to what would bemeasured in the absence of such agonist or antagonist.

[0129] Determination of whether a particular molecule inhibiting bindingof the labeled ligand to the receptor is an antagonist or an agonist isthen determined in a second, functional assay. The functionality ofGalR3 agonists and antagonists identified in the binding assay can bedetermined in cellular and animal models.

[0130] In cellular models, parameters for intracellular activitiesmediated by galanin recetors can be monitored for antgonistic and/oragonistic activities. Such parameters include but are not limited tointracellular second messenger pathways activated via the GalR3receptors, changes in cell growth rate, secretion of horemones, etc.,using published methods. Examples of the methods are measurement of theeffects of the ligands on receptor-mediated inhibition offorskolin-stimulated intracellular cAMP production [Parker et al., Mol.Brain Res. 34:179-189 (1995)], receptor-stimulated Ca⁺⁺ mobilization andmitogenic effects [Sethi et al., Cancer Res. 51:1674-1679 (1991)], andreceptor-mediated glucose-stimulated insulin release [Yanaihara et al.,Regulatory Peptides 46:93-101 (1993)].

[0131] In animal models, physiological effects of the agonists andantagonists can be evaluated by feeding the compounds and observingchanges in feeding behavior and body weight [Crawley et al., Brain Res.600:268-272 (1993)], acetylcholine release [Ogren et al., Eur. J.Pharmacology 242:59-64 (1993)], learning [Ogren et al., Neuroscience51:1-5 (1992)], memory [Robinsin et al., Behav. Neurosci. 107:458-467(1993)], and pain modulation [Verge et al., Neuroscience Letters149:193-197 (1993)].

[0132] Other Mammalian GalR3 Receptors

[0133] The present invention provides methods for cloning mammalianGalR3 receptors from other mammalian species. Briefly, Southern andNorthern blot analysis can be carried out to identify cells from otherspecies expressing genes encoding the GalR3 receptors. Complementary DNA(cDNA) libraries can be prepared by standard methods from mRNA isolatedfrom such cells, and degenerate probes or PCR primers based on thenucleic acid and amino acid sequences provided herein can be used toidentify clones encoding a GalR3 receptor.

[0134] Alternatively, expression cloning methodology can be used toidentify particular clones encoding a GalR3 receptor. An antibodypreparation which exhibits cross-reactivity with GalR3 receptors from anumber of mammalian species may be useful in monitoring expressioncloning.

[0135] However identified, clones encoding GalR3 receptors from variousmammalian species can be isolated and sequenced, and the coding regionscan be excised and inserted into an appropriate vector.

EXAMPLES

[0136] The present invention can be illustrated by the followingexamples. Unless otherwise indicated, percentages given below for solidsin solid mixtures, liquids in liquids, and solids in liquids are on awt/wt, vol/vol and wt/vol basis, respectively. Sterile conditions weregenerally maintained during cell culture.

[0137] Materials and General Methods

[0138]¹²⁵I-porcine galanin (2200 Ci/mmol) was purchased from DuPont-NEN(Boston, Mass.). Various PCR/RACE oligonucleotide primers were customsynthesized by BRL Life Technologies (Grand Island, N.Y.). MARATHON RACEcDNA was obtained from Clontech, Palo Alto, Calif. Rat galanin and C7[Brain Research 600:268-272 (1993)] were purchased from PeninsulaLaboratories (Belmont, Calif.). Rat galanin92-29) and rat galanin(3-29)were custom synthesized by Bio-synthesis, Inc.

[0139] Cloning vector pCR®2.1 and expression vector pCR®3.1 wereobtained from Invitrogen, San Diego, Calif. and used according to themanufacturer's instructions. TA cloning was carried out using pCR®2.1,whereby PCR products were ligated into the prepared vector without priorrestriction cleavage. Expression vector pCR®3.1 containing cDNA encodingthe mammalian GalR3 receptors was used to transfect COS cells. Humanbrain cDNA and human genomic DNA were from Clontech and Premega,respectively.

[0140] Standard methods were used, as described, e.g., in Maniatis etal., Molecular Cloning: A Laboratory Manual, 1982, Cold Spring HarborLaboratory, Cold Spring Harbor Press; Sambrook et al., MolecularCloning: A Laboratory Manual, (2d ed.), Vols 1-3, 1989, Cold SpringHarbor Press, NY; Ausubel et al., Biology, Greene Publishing Associates,Brooklyn, N.Y.; or Ausubel, et al. (1987 and Supplements), CurrentProtocols in Molecular Biology, Greene/Wiley, New York; Innis et al.(eds.) PCR Protocols: A Guide to Methods and Applications, 1990,Academic Press, N.Y.

[0141] The polymerase chain reaction (PCR) and rapid amplification ofcDNA ends (RACE) were carried out using the Clontech protocols. Briefly,PCR was always run with KLENTAQ polymerase, which possesses proofreading activity (Clontech), and a cycling profile of 94° C. for 1minute, 65° C. for 1 minute and 72° C. for 2 minutes (40 cycles).Approximately 1 μl of overnight E. coli cell culture was used in the PCRfor sib selection.

[0142] For RACE, nested primers specific to the rat or human GalR3 cDNAand nested adaptor primers were used in the primary and secondary PCRswith about 0.1 μg of genomic or cDNA as a template. Thermal cycling at94° C. for 30 seconds, 65° C. for 30 seconds and 72° C. for 90 seconds(25 cycles) was used in primary PCR. Cycling at 94° C. for 1 minute and70° C. for 4 minutes (30 cycles) using 5 μl of the primary PCR product(diluted 1:50) as a template was used in the secondary PCR. A GC meltreagent (Clontech) at recommended dilution was always used in both PCRand RACE reactions.

[0143] DNA sequencing was performed with ABI Prism dye termination DNAsequencing reagents and an ABI automated sequencing apparatus (PerkinElmer, Branchburg, N.J.) or manually with a SEQUITHERM ECCEL sequencingkit (Epicentre Technologies, Madison, Wis.). DNA and protein sequencecomparisons were performed with DNA* software from DNAstar Inc.,Madison, Wis.

[0144] A rat hypothalamous cDNA library was constructed by standardmethods. Briefly, total RNA from rat hypothalamous was extracted withTri-reagent-RNA/DNA/protein Isolation Reagent (Molecular ResearchCenter, Cincinnati, Ohio. Poly(A)⁺ RNA from the total RNA was purifiedwith an mRNA purification kit employing oligo(dT)-cellulosechromatography (Pharmacia, Piscataway, N.J.). Double-stranded cDNA wassynthesized from the poly(A)⁺ RNA with a Marathon cDNA amplification kit(Clontech). A portion of the cDNA was blunt-end ligated with an adaptorcontaining a BstXI restriction site. The BstXI adaptor-linked cDNA wasthen ligated into a pCDNA3 vector predigested with BstXI.

[0145] Transfection was carried out as follows. Confluent COS-7 cells(ATCC CRL 1651) grown in DMEM supplemented with 10% fetal calf serum(FCS) with 100 units/ml penicillin and 100 μg/m1 streptomycin were split1:6 into 150 mm dishes (Nunc) three days prior to transfection. On theday of transfection, the cells were approximately 90% confluent andtrypsinized off plates and washed two times with PBS without Mg⁺⁺ andCa⁺⁺. The cells were resuspended in Krebs Ringer's buffer at a densityof approximately 1.2×10⁷ cells/ml. Twenty μg of vector pCR®3.1containing rat GalR3 cDNA was diluted in Krebs Ringer's buffer (100 μlfinal volume), mixed with 0.7 ml of the COS-7 cells in a 0.4 cmelectroporation cuvettes (Bio-rad, Hercules, Calif.) then chilled on icefor 5 min. The cells were electroporated at 960 μF×260 volts (timeconstant approximately 18) followed by incubation on ice for 10 min. Thecells were incubated in DMEM with 10% FCS in a 150-mm plate.

[0146] Methods utilizing the Lipofectamine reagents (BRL LifeTechnologies) and the SuperFect reagents (Qiagene Inc., Chatsworth,Calif.) to transfect COS-7 cells worked equally well.

Example 1 Cloning and Characterization of the Rat GalR3 Receptor

[0147] In a BLAST [Altschul et al., J. Mol. Biol. 215:403-410 (1990)]search of the GenBank data base with the human GalR1 receptor amino acidsequence (Habert-Ortoli et al., supra) as a query sequence, a portion ofa clone having Accession No. Z82241 was found to possess high homologywith the human GalR1 sequence. The clone, identified as J81I2, is ahuman genomic sequence partially sequenced and arranged in the databaseas 31 segments separated by thirty 800-n sequences in random order.Amino acid residues 64-132 of the human GalR1 receptor aligned with anamino acid sequence translated at the third reading frame on thepositive strand of clone J81I2 with 55% identity. A smaller homologousmatch between amino acid residues 37-62 of the human GalR1 sequence andpart of the translated nucleotide sequence of clone J81I2 (third readingframe on the positive strand) was also found to be 57% identical in thesame analysis.

[0148] The homology level (55-57%) found with this clone issignificantly higher than a 40% homology found between the GalR1 andGalR2 receptors, and markedly lower than those between the specieshomologs of GalR1 receptors among human, rat and mouse (>90%). Thus,part of the nucleotide sequence of clone J81I2 appeared to encode anamino acid sequence, corresponding to amino acid residues 37-132 ofhuman GalR1, of a new human galanin receptor designated the human GalR3receptor.

[0149] In brief, the strategy used to obtain cDNA encoding the rat GalR3receptor was to generate several pairs of PCR primers, based on thehuman genomic clone, and to use them in RT-PCR with rat hypothalamus RNAas template to obtain a cDNA sequence of the GalR3 cDNA.

[0150] Two PCR primers, designated oligo93C (SEQ ID NO: 5) and oligo120B(SEQ ID NO: 6), produced a PCR product of approximately 700 bp which wascloned into vector pCR®3.1. The DNA sequence of the clone was comparedwith the nucleotide sequences in GenBank and the search resultsrevealed, in rank order, the human genomic clone (Z82241), rGalR2, andhGalR1 as the most homologous sequences, with identities of 86%, 65% and63%, respectively. The rat clone thus appeared to be the species homologof the putative human GalR3 cDNA.

[0151] To extend the cDNA sequence toward the 5′ and 3′ directions, RACEand PCR sib selection were used. In RACE amplification, primersdesignated oligo172 (SEQ ID NO: 7) and API (SEQ ID NO: 8; outer adaptorprimer) were used in the primary PCR and others designated oligo177 (SEQID NO: 9) and AP2 (SEQ ID NO: 10; inner adaptor primer) were used in thesecondary PCR. The final RACE product, ≈1.8 kb, contained part of the 5′end of rat GalR3 cDNA and the upstream 5′ untranslated region.

[0152] In PCR sib selection, two primers designated oligo164 (SEQ ID NO:11) and oligo167 (SEQ ID NO: 12) were used to screen a cDNA libraryconstructed from rat hypothalamus. The library was pooled at a size of≈5000 clones/pool, and DNA was prepared for each of the individualpools. A pool designated A28 gave a positive band amplified with the twoprimers and was sub-divided and screened until a single clone wasobtained. That clone, designated A28-1, was 1.3 kb long and possessedmost of the putative rat GalR3 cDNA.

[0153] A full-length rat GAlR3 cDNA clone was obtained by performingfurther sib selection on the rat hypothalamus cDNA library using primeroligo185 (SEQ ID NO: 13), based on the sequence of the 5′ RACE product,and primer oligo184 (SEQ ID NO: 14), based on the sequence of cloneA28-1. A single clone A5-3 selected from library pool A5 was obtainedand sequenced. That clone was 2.2 kb long and contained all of thesequence of clone A28-1 and part of the 5′ RACE product. A complete openreading frame (ORF) was identified in the clone, and the deduced aminoacid sequence of the ORF consisted of 370 amino acids with a calculatedmolecular mass of 40.3 kDa.

[0154] Hydropathy analysis and comparison with other GalR receptorsrevealed seven putative transmembrane spanning domains (TMs) typical ofG-protein coupled receptors. The GalR3 receptor also contains a singlepotential N-linked glycosylation site in the N-terminal region, two Cysresidues in the first and second extracellular loops that form aputative disulfide bond in these receptors, and two Cys residues in theC-terminal region that may be involved in palmitoylation.

Example 2 Cloning and Characterization of the Human GalR3 Receptor

[0155] To obtain the cDNA of the human GalR3 receptor, primersdesignated oligo 93 (SEQ ID NO: 15) and oligo 94 (SEQ ID NO: 16) basedon the human GalR3 nucleotide sequence (Z82241) were used in PCR togenerate the fragment with cDNA prepared by reverse-transcription ofhuman brain cDNA (Clontech; 0.1 μg) and human placenta genomic DNA(Premega; 0.1 μg) as template. A single band PCR product of 300 bp inlength, as analyzed with agarose gel electrophoresis, was generated inboth PCR reactions. The PCR product obtained with genomic DNA astemplate was cloned into the pCR®2.1 vector to produce a clonedesignated pCR2.1-f93/94, and sequencing analysis showed that the clonedfragment was identical to part of the putative GalR3 sequence in genomicclone J81I2.

[0156] A series of nested forward and reverse primers within thissequence and human brain cDNA linked with adapters at the two ends wasthen used in RACE PCR to obtain the upstream and downstream cDNAsequences covering the start and stop codons. A 5′-RACE product, 0.5 kblong, was obtained with oligo 134 (SEQ ID NO: 17) and oligo 135 (SEQ IDNO: 18) as the nested GalR3-specific primers and the twoadaptor-specific primers AP1 (SEQ ID NO: 8) and AP2 (SEQ ID NO: 10).Similarly, an 1-kb 3′-RACE product was obtained with oligo 93B (SEQ IDNO: 19) and oligo 93C (SEQ ID NO: 5) as the nested GalR3-specificprimers. Sequencing of these two fragments revealed an in-frame startcodon in the 5′-RACE product and an in-frame stop codon in the 3′-RACEproduct.

[0157] The full length cDNA of the GalR3 receptor was obtained by PCRusing human brain cDNA as template and primers designated oligo 154 (SEQID NO: 20) and oligo 159A (SEQ ID NO: 21) to produce the long form, andprimers designated oligo 156 (SEQ ID NO: 22) and oligo 159A (SEQ ID NO:21) to produce the short form of the human GalR3 receptor.

Example 3 Agonist/Antagonist Screening Assay

[0158] Receptor membranes were prepared as follows. COS-7 cellstransfected as described above with vector pCR®3.1 containing cDNAencoding the rat GalR3 receptor were incubated in DMEM with 10% FCS in150 mm plates for 3 days in a humidified 5% CO₂ incubator, after whichthe medium was removed and the cells were washed three times withphosphate buffered saline (PBS).

[0159] To each plate, 5 ml of 5 mM Hepes buffer (pH 7.4), 0.1 mM PMSF,and 0.1 mg/ml bacitracin were added and incubated at room temperaturefor 15 minutes. The cells were scraped from the plates and centrifugedat 13,000×g for 15 minutes at 4° C. The resulting cell pellet wasresuspended in 2 ml of 25 mM Tris-Cl (pH7.4) containing 0.2 mM PMSF byvortexing, and dispersed with a syringe attached with a #23 gaugeneedle. Protein concentrations were determined using a BCA(bicinchoninic acid) method (Pierce, Rockford, Ill.).

[0160] Binding of ¹²⁵I-porcine-galanin to the membrane preparations wasperformed in a buffer containing 25 mM Tris-Cl (pH7.4), 1% bovine serumalbumin (w/v), 0.1% bacitracin, 2 μg/ml leupeptin and 10 mM MgCl₂.Ligand saturation plots were performed using 20 μg amounts of themembrane protein in a total volume of 200 μl using 3 μM cold galanin todetermine nonspecific binding. Peptide competition studies wereperformed in a total volume of 200 μl, containing 20 μg of membraneprotein and 0.3 nM ¹²⁵I-porcine galanin. Incubations were carried out atroom temperature for 1 hour and were terminated by rapid vacuumfiltration through MULTISCREEN FB (glass fiber B) Filter Plates(Millipore, Bedford, Mass.) which had been pre-treated with 0.3%polyethylenimine to prevent non-specific binding of the radioligand tothe filter. The filters were then washed three times with 100 μl of PBS(pH7.4). All data were analyzed using non-linear regression software(Prism, GraphPad, San Diego, Calif.), and the Ki was calculated usingthe method of Cheng and Prusoff [Biochem. Pharmacol. 22:3099-3108(1973)].

[0161] In a typical assay, the results of which are shown in FIG. 2, thecompeting ligands rat galanin (), chimeric galanin peptide C7 (◯),galanin(2-29) (□), galanin(3-29) (Δ) and galanin(10-20) (▴) produced Kivalues of 1.2, 1.4, 14, >1,000 and >1,000 nM, respectively. The curvesshown indicate the fits of the data points by nonlinear regression forone-site binding.

[0162] As shown in FIG. 2, compounds known to have galanin activity,i.e., galanin itself, galanin peptide C7 and galanin(2-29), were allcompetitive inhibitors of the labeled galanin, whereas the compoundslacking galanin activity, i.e., galanin(3-29) and galanin(10-20), werenot. Thus, the assay has a high degree of ligand specificity and shouldbe generally applicable to the identification of galanin agonists andantagonists.

[0163] Many modifications and variations of this invention can be madewithout departing from its spirit and scope, as will be apparent tothose skilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims, together with the full scope ofequivalents to which such claims are entitled.

1 22 1113 base pairs nucleic acid double linear 1 ATG GCT GAC ATC CAGAAC ATT TCG CTG GAC AGC CCA GGG AGC GTA GGG 48 Met Ala Asp Ile Gln AsnIle Ser Leu Asp Ser Pro Gly Ser Val Gly 1 5 10 15 GCT GTG GCA GTG CCTGTG ATC TTT GCC CTC ATC TTC CTG TTG GGC ATG 96 Ala Val Ala Val Pro ValIle Phe Ala Leu Ile Phe Leu Leu Gly Met 20 25 30 GTG GGC AAT GGG CTG GTGTTG GCT GTG CTA CTG CAG CCT GGC CCA AGT 144 Val Gly Asn Gly Leu Val LeuAla Val Leu Leu Gln Pro Gly Pro Ser 35 40 45 GCC TGG CAG GAG CAA GGG AGTACA CAA GAT CTC TTC ATC CTC AAC TTG 192 Ala Trp Gln Glu Gln Gly Ser ThrGln Asp Leu Phe Ile Leu Asn Leu 50 55 60 GCC GTG GCC GAC CTT TGC TTC ATCCTG TGC TGC GTG CCC TTC CAG GCA 240 Ala Val Ala Asp Leu Cys Phe Ile LeuCys Cys Val Pro Phe Gln Ala 65 70 75 80 GCC ATC TAC ACC CTG GAT GCC TGGCTC TTT GGG GCT TTC GTG TGC AAG 288 Ala Ile Tyr Thr Leu Asp Ala Trp LeuPhe Gly Ala Phe Val Cys Lys 85 90 95 ACG GTA CAT CTG CTC ATC TAC CTC ACCATG TAT GCC AGC AGC TTC ACC 336 Thr Val His Leu Leu Ile Tyr Leu Thr MetTyr Ala Ser Ser Phe Thr 100 105 110 CTG GCG GCC GTC TCC CTG GAC AGG TACCTG GCT GTG CGG CAC CAA CTG 384 Leu Ala Ala Val Ser Leu Asp Arg Tyr LeuAla Val Arg His Gln Leu 115 120 125 CGC TCC AGA GCC CTG CGC ACC CCG TGCAAC GCG CGC GCC GCC GTG GGG 432 Arg Ser Arg Ala Leu Arg Thr Pro Cys AsnAla Arg Ala Ala Val Gly 130 135 140 CTC GTG TGG CTG CTG GGG GCT CTC TTTTCC GCG CCC TAC CTA AGC TAC 480 Leu Val Trp Leu Leu Gly Ala Leu Phe SerAla Pro Tyr Leu Ser Tyr 145 150 155 160 TAC GGC ACG GTG CGC TAC GGC GCGCTC GAG CTC TGC GTG CCC GCT TTG 528 Tyr Gly Thr Val Arg Tyr Gly Ala LeuGlu Leu Cys Val Pro Ala Leu 165 170 175 GAG GAC GCG CGG CGG CGG CGC TTGGAC GTG GCC GCC TTC GCC GCG GGC 576 Glu Asp Ala Arg Arg Arg Arg Leu AspVal Ala Ala Phe Ala Ala Gly 180 185 190 TAC CTG CTG CCG GTG GCC GTG GTGAGC CTG GCC TAC GGA CGC ACG CTA 624 Tyr Leu Leu Pro Val Ala Val Val SerLeu Ala Tyr Gly Arg Thr Leu 195 200 205 TGT CTT CTA TGG GCC GCC GTG GGTCCC GCG GGC GCG GCG GCA GCA GAG 672 Cys Leu Leu Trp Ala Ala Val Gly ProAla Gly Ala Ala Ala Ala Glu 210 215 220 GCG CGC AGA CGG GCG ACC GGC CGGGCG GGA CGG GCC ATG CTG GCA GTG 720 Ala Arg Arg Arg Ala Thr Gly Arg AlaGly Arg Ala Met Leu Ala Val 225 230 235 240 GCC GCG CTC TAC GCG CTT TGCTGG GGC CCG CAC CAC GCG CTC ATC CTC 768 Ala Ala Leu Tyr Ala Leu Cys TrpGly Pro His His Ala Leu Ile Leu 245 250 255 TGC TTC TGG TAC GGT CGG TTCGCC TTC AGC CCG GCC ACC TAC GCC ATT 816 Cys Phe Trp Tyr Gly Arg Phe AlaPhe Ser Pro Ala Thr Tyr Ala Ile 260 265 270 CGC CTG GCC TCG CAC TGC CTCGCC TAC GCC AAC TCC TGC CTT AAC CCG 864 Arg Leu Ala Ser His Cys Leu AlaTyr Ala Asn Ser Cys Leu Asn Pro 275 280 285 CTC GTC TAC TCG CTC GCC TCGCGC CAC TTC CGC GCG CGC TTC CGC CGC 912 Leu Val Tyr Ser Leu Ala Ser ArgHis Phe Arg Ala Arg Phe Arg Arg 290 295 300 CTG TGG CCC TGC GGC CGT TGCCGC CAC CGC CAC CAC CAC CGC GCT CAT 960 Leu Trp Pro Cys Gly Arg Cys ArgHis Arg His His His Arg Ala His 305 310 315 320 CGA GCC CTC CGT CGT GTCCAG CCG GCG TCT TCG GGC CCC GCC GGT TAT 1008 Arg Ala Leu Arg Arg Val GlnPro Ala Ser Ser Gly Pro Ala Gly Tyr 325 330 335 CCC GGC GAC GCC AGG CCTCGT GGT TGG AGT ATG GAG CCC AGA GGG GAT 1056 Pro Gly Asp Ala Arg Pro ArgGly Trp Ser Met Glu Pro Arg Gly Asp 340 345 350 GCT CTG CGT GGT GGT GGAGAG ACT AGA CTA ACC CTG TCC CCC AGG GGA 1104 Ala Leu Arg Gly Gly Gly GluThr Arg Leu Thr Leu Ser Pro Arg Gly 355 360 365 CCT CAA TAA 1113 Pro Gln370 370 amino acids amino acid linear 2 Met Ala Asp Ile Gln Asn Ile SerLeu Asp Ser Pro Gly Ser Val Gly 1 5 10 15 Ala Val Ala Val Pro Val IlePhe Ala Leu Ile Phe Leu Leu Gly Met 20 25 30 Val Gly Asn Gly Leu Val LeuAla Val Leu Leu Gln Pro Gly Pro Ser 35 40 45 Ala Trp Gln Glu Gln Gly SerThr Gln Asp Leu Phe Ile Leu Asn Leu 50 55 60 Ala Val Ala Asp Leu Cys PheIle Leu Cys Cys Val Pro Phe Gln Ala 65 70 75 80 Ala Ile Tyr Thr Leu AspAla Trp Leu Phe Gly Ala Phe Val Cys Lys 85 90 95 Thr Val His Leu Leu IleTyr Leu Thr Met Tyr Ala Ser Ser Phe Thr 100 105 110 Leu Ala Ala Val SerLeu Asp Arg Tyr Leu Ala Val Arg His Gln Leu 115 120 125 Arg Ser Arg AlaLeu Arg Thr Pro Cys Asn Ala Arg Ala Ala Val Gly 130 135 140 Leu Val TrpLeu Leu Gly Ala Leu Phe Ser Ala Pro Tyr Leu Ser Tyr 145 150 155 160 TyrGly Thr Val Arg Tyr Gly Ala Leu Glu Leu Cys Val Pro Ala Leu 165 170 175Glu Asp Ala Arg Arg Arg Arg Leu Asp Val Ala Ala Phe Ala Ala Gly 180 185190 Tyr Leu Leu Pro Val Ala Val Val Ser Leu Ala Tyr Gly Arg Thr Leu 195200 205 Cys Leu Leu Trp Ala Ala Val Gly Pro Ala Gly Ala Ala Ala Ala Glu210 215 220 Ala Arg Arg Arg Ala Thr Gly Arg Ala Gly Arg Ala Met Leu AlaVal 225 230 235 240 Ala Ala Leu Tyr Ala Leu Cys Trp Gly Pro His His AlaLeu Ile Leu 245 250 255 Cys Phe Trp Tyr Gly Arg Phe Ala Phe Ser Pro AlaThr Tyr Ala Ile 260 265 270 Arg Leu Ala Ser His Cys Leu Ala Tyr Ala AsnSer Cys Leu Asn Pro 275 280 285 Leu Val Tyr Ser Leu Ala Ser Arg His PheArg Ala Arg Phe Arg Arg 290 295 300 Leu Trp Pro Cys Gly Arg Cys Arg HisArg His His His Arg Ala His 305 310 315 320 Arg Ala Leu Arg Arg Val GlnPro Ala Ser Ser Gly Pro Ala Gly Tyr 325 330 335 Pro Gly Asp Ala Arg ProArg Gly Trp Ser Met Glu Pro Arg Gly Asp 340 345 350 Ala Leu Arg Gly GlyGly Glu Thr Arg Leu Thr Leu Ser Pro Arg Gly 355 360 365 Pro Gln 370 1275base pairs nucleic acid double linear 3 ATG ACT TTG GCT CTG CTC TCC CCTCCT CCA TCT CCC ACG AGC TTC CAG 48 Met Thr Leu Ala Leu Leu Ser Pro ProPro Ser Pro Thr Ser Phe Gln 1 5 10 15 CCC AGA ACA CCT GGC CAG ACC CAGGTC GGG GGA GTT AGA TCC CGG GGT 96 Pro Arg Thr Pro Gly Gln Thr Gln ValGly Gly Val Arg Ser Arg Gly 20 25 30 CAA GCA ACC AGA ACT GGG GGC TCT TGCCTG AGG ATT CCA GCT TCT CTT 144 Gln Ala Thr Arg Thr Gly Gly Ser Cys LeuArg Ile Pro Ala Ser Leu 35 40 45 CCC AGG TGC CCG TCT GAT GGG GAG ATG GCTGAT GCC CAG AAC ATT TCA 192 Pro Arg Cys Pro Ser Asp Gly Glu Met Ala AspAla Gln Asn Ile Ser 50 55 60 CTG GAC AGC CCA GGG AGT GTG GGG GCC GTG GCAGTG CAT GTG GTC TTT 240 Leu Asp Ser Pro Gly Ser Val Gly Ala Val Ala ValHis Val Val Phe 65 70 75 80 GCC CTA ATC TTC CTG CTG GGC ACA GTG GGC AATGGG CTG GTG CTG GCA 288 Ala Leu Ile Phe Leu Leu Gly Thr Val Gly Asn GlyLeu Val Leu Ala 85 90 95 GTG CTC CTG CAG CCT GGC CCG AGT GCC TGG CAG GAGCCT TGC AGC ACC 336 Val Leu Leu Gln Pro Gly Pro Ser Ala Trp Gln Glu ProCys Ser Thr 100 105 110 ACG GAC CTG TTC ATC CTC AAC CTG GCG GTG GCT GACCTC TGC TTC ATC 384 Thr Asp Leu Phe Ile Leu Asn Leu Ala Val Ala Asp LeuCys Phe Ile 115 120 125 CTG TGC TGC GTG CCC TTC CAA GCC ACC ATC TAC ACGCTG GAT GCC TGG 432 Leu Cys Cys Val Pro Phe Gln Ala Thr Ile Tyr Thr LeuAsp Ala Trp 130 135 140 CTC TTT GGG GCC CTC GTC TGC AAC GCC GTG CAC CTGCTC ATC TAC CTC 480 Leu Phe Gly Ala Leu Val Cys Asn Ala Val His Leu LeuIle Tyr Leu 145 150 155 160 ACC ATG TAC GCC AGC AGC TTT ACG CTG GCT GCTGTC TCC GTG GAC AGG 528 Thr Met Tyr Ala Ser Ser Phe Thr Leu Ala Ala ValSer Val Asp Arg 165 170 175 TAC CTG GCC GTG CGG CAC CCG CTG CGC TCG CGCGCC CTG CGC ACG CCG 576 Tyr Leu Ala Val Arg His Pro Leu Arg Ser Arg AlaLeu Arg Thr Pro 180 185 190 CGT AAC GCC CGC GCC GCA GTG GGG CTG GTG TGGCTG CTG GCG GCG CTC 624 Arg Asn Ala Arg Ala Ala Val Gly Leu Val Trp LeuLeu Ala Ala Leu 195 200 205 TTC TCG GCG CCC TAC CTC AGC TAC TAC GGC ACCGTG CGC TAC GGC GCG 672 Phe Ser Ala Pro Tyr Leu Ser Tyr Tyr Gly Thr ValArg Tyr Gly Ala 210 215 220 CTG GAG CTC TGC GTG CCC GCC TGG GAG GAC GCGCGC CGC CGC GCC CGG 720 Leu Glu Leu Cys Val Pro Ala Trp Glu Asp Ala ArgArg Arg Ala Arg 225 230 235 240 GAC GTG GCC ACC TAC GCT GCC GGC TAC CTGCTG CCC GTG GCC GTG GTG 768 Asp Val Ala Thr Tyr Ala Ala Gly Tyr Leu LeuPro Val Ala Val Val 245 250 255 AGC CTG GCC TAC GGG CGC ACG CTG CGC TTCCTG TGG GCC GCC GTG GGT 816 Ser Leu Ala Tyr Gly Arg Thr Leu Arg Phe LeuTrp Ala Ala Val Gly 260 265 270 CCC GCG GGC GCG GCG GCG GCC GAG GCG CGGCGG AGG GCG ACG GGC CGC 864 Pro Ala Gly Ala Ala Ala Ala Glu Ala Arg ArgArg Ala Thr Gly Arg 275 280 285 GCG GGG CGC GCC ATG CTG GCG GTG GCC GCGCTC TAC GCG CTC TGC TGG 912 Ala Gly Arg Ala Met Leu Ala Val Ala Ala LeuTyr Ala Leu Cys Trp 290 295 300 GGT CCG CAC CAC GCG CTC ATC CTG TGC TTCTGG TAC GGC CGC TTC GCC 960 Gly Pro His His Ala Leu Ile Leu Cys Phe TrpTyr Gly Arg Phe Ala 305 310 315 320 TTC AGC CCG GCC ACC TAC GCC TGC CGCCTG GCC TCA CAC TGC CTG GCC 1008 Phe Ser Pro Ala Thr Tyr Ala Cys Arg LeuAla Ser His Cys Leu Ala 325 330 335 TAC GCC AAC TCC TGC CTC AAC CCG CTCGTC TAC GCG CTC GCC TCG CGC 1056 Tyr Ala Asn Ser Cys Leu Asn Pro Leu ValTyr Ala Leu Ala Ser Arg 340 345 350 CAC TTC CGC GCG CGC TTC CGC CGC CTGTGG CCG TGC GGC CGC CGA CGC 1104 His Phe Arg Ala Arg Phe Arg Arg Leu TrpPro Cys Gly Arg Arg Arg 355 360 365 CGC CAC CGT GCC CGC CGC GCT TTG CGTCGC GTC CGC CCC GCG TCC TCG 1152 Arg His Arg Ala Arg Arg Ala Leu Arg ArgVal Arg Pro Ala Ser Ser 370 375 380 GGC CCA CCC GGC TGC CCC GGA GAC GCCCGG CCT AGC GGG GGG CTG CTG 1200 Gly Pro Pro Gly Cys Pro Gly Asp Ala ArgPro Ser Gly Gly Leu Leu 385 390 395 400 GCT GGT GGC GGC CAG GGC CCG GAGCCC AGG GAG GGA CCC GTC CAC GGC 1248 Ala Gly Gly Gly Gln Gly Pro Glu ProArg Glu Gly Pro Val His Gly 405 410 415 GGA GAG GCT GCC CGA GGA CCG GAATAA 1275 Gly Glu Ala Ala Arg Gly Pro Glu 420 424 amino acids amino acidlinear 4 Met Thr Leu Ala Leu Leu Ser Pro Pro Pro Ser Pro Thr Ser Phe Gln1 5 10 15 Pro Arg Thr Pro Gly Gln Thr Gln Val Gly Gly Val Arg Ser ArgGly 20 25 30 Gln Ala Thr Arg Thr Gly Gly Ser Cys Leu Arg Ile Pro Ala SerLeu 35 40 45 Pro Arg Cys Pro Ser Asp Gly Glu Met Ala Asp Ala Gln Asn IleSer 50 55 60 Leu Asp Ser Pro Gly Ser Val Gly Ala Val Ala Val His Val ValPhe 65 70 75 80 Ala Leu Ile Phe Leu Leu Gly Thr Val Gly Asn Gly Leu ValLeu Ala 85 90 95 Val Leu Leu Gln Pro Gly Pro Ser Ala Trp Gln Glu Pro CysSer Thr 100 105 110 Thr Asp Leu Phe Ile Leu Asn Leu Ala Val Ala Asp LeuCys Phe Ile 115 120 125 Leu Cys Cys Val Pro Phe Gln Ala Thr Ile Tyr ThrLeu Asp Ala Trp 130 135 140 Leu Phe Gly Ala Leu Val Cys Asn Ala Val HisLeu Leu Ile Tyr Leu 145 150 155 160 Thr Met Tyr Ala Ser Ser Phe Thr LeuAla Ala Val Ser Val Asp Arg 165 170 175 Tyr Leu Ala Val Arg His Pro LeuArg Ser Arg Ala Leu Arg Thr Pro 180 185 190 Arg Asn Ala Arg Ala Ala ValGly Leu Val Trp Leu Leu Ala Ala Leu 195 200 205 Phe Ser Ala Pro Tyr LeuSer Tyr Tyr Gly Thr Val Arg Tyr Gly Ala 210 215 220 Leu Glu Leu Cys ValPro Ala Trp Glu Asp Ala Arg Arg Arg Ala Arg 225 230 235 240 Asp Val AlaThr Tyr Ala Ala Gly Tyr Leu Leu Pro Val Ala Val Val 245 250 255 Ser LeuAla Tyr Gly Arg Thr Leu Arg Phe Leu Trp Ala Ala Val Gly 260 265 270 ProAla Gly Ala Ala Ala Ala Glu Ala Arg Arg Arg Ala Thr Gly Arg 275 280 285Ala Gly Arg Ala Met Leu Ala Val Ala Ala Leu Tyr Ala Leu Cys Trp 290 295300 Gly Pro His His Ala Leu Ile Leu Cys Phe Trp Tyr Gly Arg Phe Ala 305310 315 320 Phe Ser Pro Ala Thr Tyr Ala Cys Arg Leu Ala Ser His Cys LeuAla 325 330 335 Tyr Ala Asn Ser Cys Leu Asn Pro Leu Val Tyr Ala Leu AlaSer Arg 340 345 350 His Phe Arg Ala Arg Phe Arg Arg Leu Trp Pro Cys GlyArg Arg Arg 355 360 365 Arg His Arg Ala Arg Arg Ala Leu Arg Arg Val ArgPro Ala Ser Ser 370 375 380 Gly Pro Pro Gly Cys Pro Gly Asp Ala Arg ProSer Gly Gly Leu Leu 385 390 395 400 Ala Gly Gly Gly Gln Gly Pro Glu ProArg Glu Gly Pro Val His Gly 405 410 415 Gly Glu Ala Ala Arg Gly Pro Glu420 25 base pairs nucleic acid single linear 5 GCTGGCAGTG CTCCTGCAGCCTGGC 25 27 base pairs nucleic acid single linear 6 AAGCGGCCGTACCAGAAGCA CAGGATG 27 26 base pairs nucleic acid single linear 7TGCGGGCCCC AGCAGAGCGC GTAGAG 26 27 base pairs nucleic acid single linear8 CCATCCTAAT ACGACTCACT ATAGGGC 27 24 base pairs nucleic acid singlelinear 9 CATCCAGTGT GTAGATGGCT GCCT 24 23 base pairs nucleic acid singlelinear 10 ACTCACTATA GGGCTCGAGC GGC 23 26 base pairs nucleic acid singlelinear 11 CCAAGTGCCT GGCAGGAGCC AAGCAG 26 28 base pairs nucleic acidsingle linear 12 CGCGTAGAGC GCGGCCACTG CCAGCATG 28 26 base pairs nucleicacid single linear 13 CAAGGGCTGA ATCAANAAGC TCCAGC 26 28 base pairsnucleic acid single linear 14 GCGGGTTAAG GCANGAGTTG GCGTAGGC 28 25 basepairs nucleic acid single linear 15 GTGGTCTTTG CCCTAATCTT CCTGC 25 25base pairs nucleic acid single linear 16 ACGGAGACAG CAGCCAGCGT AAAGC 2525 base pairs nucleic acid single linear 17 CTTGGAAGGG CACGCAGCAC AGGAT25 25 base pairs nucleic acid single linear 18 GCCGCAGAGG TCAGCCACCGCCAGG 25 25 base pairs nucleic acid single linear 19 TGGGCACAGTGGGCAATGGG CTGGT 25 25 base pairs nucleic acid single linear 20GAAACTGAGG AACTCTCACC CCTTG 25 25 base pairs nucleic acid single linear21 TTGCAGGCGG CAGGGTTTAT TCCGG 25 26 base pairs nucleic acid singlelinear 22 CAAGCAACCA GAACTGGGGG CTCTTG 26 SEQUENCE LISTING (1) GENERALINFORMATION 1 (i) APPLICANT Bayne, Marvin Hashemi, Tanaz He, ChaogangWang, Suke (ii) TITLE OF INVENTION Mammalian Galanin Receptors (iii)NUMBER OF SEQUENCES 22 (iv) CORRESPONDENCE ADDRESS (A) ADDRESSEESchering-Plough Corporation (B) STREET 2000 Galloping Hill Road 2000 (C)CITY Kenilworth (D) STATE New Jersey (E) COUNTRY USA (F) ZIP 07033-0530070330530 (v) COMPUTER READABLE FORM (A) MEDIUM TYPE Floppy disk (B)COMPUTER Power Macintosh 7600/120 7600120 (C) OPERATING SYSTEM Macintosh7.5.3 753 (D) SOFTWARE Microsoft Word 6.0 60 (vi) CURRENT APPLICATIONDATA (A) APPLICATION NUMBERUS/10/779,021 (B) FILING DATE 12-FEB-2004122004 (C) CLASSIFICATION (vii) PRIOR APPLICATION DATA (A) APPLICATIONNUMBERUS/08/916,247 08916247 (B) FILING DATE 22-AUG-1997 221997 (C)CLASSIFICATION (viii) ATTORNEY/AGENT INFORMATION (A) NAME Dulak, NormanC. (B) REGISTRATION NUMBER 31,608 31608 (C) REFERENCE/DOCKET NUMBERCN0769 0769 (ix) TELECOMMUNICATION INFORMATION (A) TELEPHONE 908 2982906 9082982906 (B) TELEFAX 908 298 5388 9082985388 (C) TELEX 1113 basepairs nucleic acid double linear 1 ATG GCT GAC ATC CAG AAC ATT TCG CTGGAC AGC CCA GGG AGC GTA GGG 48 Met Ala Asp Ile Gln Asn Ile Ser Leu AspSer Pro Gly Ser Val Gly 1 5 10 15 GCT GTG GCA GTG CCT GTG ATC TTT GCCCTC ATC TTC CTG TTG GGC ATG 96 Ala Val Ala Val Pro Val Ile Phe Ala LeuIle Phe Leu Leu Gly Met 20 25 30 GTG GGC AAT GGG CTG GTG TTG GCT GTG CTACTG CAG CCT GGC CCA AGT 144 Val Gly Asn Gly Leu Val Leu Ala Val Leu LeuGln Pro Gly Pro Ser 35 40 45 GCC TGG CAG GAG CAA GGG AGT ACA CAA GAT CTCTTC ATC CTC AAC TTG 192 Ala Trp Gln Glu Gln Gly Ser Thr Gln Asp Leu PheIle Leu Asn Leu 50 55 60 GCC GTG GCC GAC CTT TGC TTC ATC CTG TGC TGC GTGCCC TTC CAG GCA 240 Ala Val Ala Asp Leu Cys Phe Ile Leu Cys Cys Val ProPhe Gln Ala 65 70 75 80 GCC ATC TAC ACC CTG GAT GCC TGG CTC TTT GGG GCTTTC GTG TGC AAG 288 Ala Ile Tyr Thr Leu Asp Ala Trp Leu Phe Gly Ala PheVal Cys Lys 85 90 95 ACG GTA CAT CTG CTC ATC TAC CTC ACC ATG TAT GCC AGCAGC TTC ACC 336 Thr Val His Leu Leu Ile Tyr Leu Thr Met Tyr Ala Ser SerPhe Thr 100 105 110 CTG GCG GCC GTC TCC CTG GAC AGG TAC CTG GCT GTG CGGCAC CAA CTG 384 Leu Ala Ala Val Ser Leu Asp Arg Tyr Leu Ala Val Arg HisGln Leu 115 120 125 CGC TCC AGA GCC CTG CGC ACC CCG TGC AAC GCG CGC GCCGCC GTG GGG 432 Arg Ser Arg Ala Leu Arg Thr Pro Cys Asn Ala Arg Ala AlaVal Gly 130 135 140 CTC GTG TGG CTG CTG GGG GCT CTC TTT TCC GCG CCC TACCTA AGC TAC 480 Leu Val Trp Leu Leu Gly Ala Leu Phe Ser Ala Pro Tyr LeuSer Tyr 145 150 155 160 TAC GGC ACG GTG CGC TAC GGC GCG CTC GAG CTC TGCGTG CCC GCT TTG 528 Tyr Gly Thr Val Arg Tyr Gly Ala Leu Glu Leu Cys ValPro Ala Leu 165 170 175 GAG GAC GCG CGG CGG CGG CGC TTG GAC GTG GCC GCCTTC GCC GCG GGC 576 Glu Asp Ala Arg Arg Arg Arg Leu Asp Val Ala Ala PheAla Ala Gly 180 185 190 TAC CTG CTG CCG GTG GCC GTG GTG AGC CTG GCC TACGGA CGC ACG CTA 624 Tyr Leu Leu Pro Val Ala Val Val Ser Leu Ala Tyr GlyArg Thr Leu 195 200 205 TGT CTT CTA TGG GCC GCC GTG GGT CCC GCG GGC GCGGCG GCA GCA GAG 672 Cys Leu Leu Trp Ala Ala Val Gly Pro Ala Gly Ala AlaAla Ala Glu 210 215 220 GCG CGC AGA CGG GCG ACC GGC CGG GCG GGA CGG GCCATG CTG GCA GTG 720 Ala Arg Arg Arg Ala Thr Gly Arg Ala Gly Arg Ala MetLeu Ala Val 225 230 235 240 GCC GCG CTC TAC GCG CTT TGC TGG GGC CCG CACCAC GCG CTC ATC CTC 768 Ala Ala Leu Tyr Ala Leu Cys Trp Gly Pro His HisAla Leu Ile Leu 245 250 255 TGC TTC TGG TAC GGT CGG TTC GCC TTC AGC CCGGCC ACC TAC GCC ATT 816 Cys Phe Trp Tyr Gly Arg Phe Ala Phe Ser Pro AlaThr Tyr Ala Ile 260 265 270 CGC CTG GCC TCG CAC TGC CTC GCC TAC GCC AACTCC TGC CTT AAC CCG 864 Arg Leu Ala Ser His Cys Leu Ala Tyr Ala Asn SerCys Leu Asn Pro 275 280 285 CTC GTC TAC TCG CTC GCC TCG CGC CAC TTC CGCGCG CGC TTC CGC CGC 912 Leu Val Tyr Ser Leu Ala Ser Arg His Phe Arg AlaArg Phe Arg Arg 290 295 300 CTG TGG CCC TGC GGC CGT TGC CGC CAC CGC CACCAC CAC CGC GCT CAT 960 Leu Trp Pro Cys Gly Arg Cys Arg His Arg His HisHis Arg Ala His 305 310 315 320 CGA GCC CTC CGT CGT GTC CAG CCG GCG TCTTCG GGC CCC GCC GGT TAT 1008 Arg Ala Leu Arg Arg Val Gln Pro Ala Ser SerGly Pro Ala Gly Tyr 325 330 335 CCC GGC GAC GCC AGG CCT CGT GGT TGG AGTATG GAG CCC AGA GGG GAT 1056 Pro Gly Asp Ala Arg Pro Arg Gly Trp Ser MetGlu Pro Arg Gly Asp 340 345 350 GCT CTG CGT GGT GGT GGA GAG ACT AGA CTAACC CTG TCC CCC AGG GGA 1104 Ala Leu Arg Gly Gly Gly Glu Thr Arg Leu ThrLeu Ser Pro Arg Gly 355 360 365 CCT CAA TAA 1113 Pro Gln 370 370 aminoacids amino acid linear 2 Met Ala Asp Ile Gln Asn Ile Ser Leu Asp SerPro Gly Ser Val Gly 1 5 10 15 Ala Val Ala Val Pro Val Ile Phe Ala LeuIle Phe Leu Leu Gly Met 20 25 30 Val Gly Asn Gly Leu Val Leu Ala Val LeuLeu Gln Pro Gly Pro Ser 35 40 45 Ala Trp Gln Glu Gln Gly Ser Thr Gln AspLeu Phe Ile Leu Asn Leu 50 55 60 Ala Val Ala Asp Leu Cys Phe Ile Leu CysCys Val Pro Phe Gln Ala 65 70 75 80 Ala Ile Tyr Thr Leu Asp Ala Trp LeuPhe Gly Ala Phe Val Cys Lys 85 90 95 Thr Val His Leu Leu Ile Tyr Leu ThrMet Tyr Ala Ser Ser Phe Thr 100 105 110 Leu Ala Ala Val Ser Leu Asp ArgTyr Leu Ala Val Arg His Gln Leu 115 120 125 Arg Ser Arg Ala Leu Arg ThrPro Cys Asn Ala Arg Ala Ala Val Gly 130 135 140 Leu Val Trp Leu Leu GlyAla Leu Phe Ser Ala Pro Tyr Leu Ser Tyr 145 150 155 160 Tyr Gly Thr ValArg Tyr Gly Ala Leu Glu Leu Cys Val Pro Ala Leu 165 170 175 Glu Asp AlaArg Arg Arg Arg Leu Asp Val Ala Ala Phe Ala Ala Gly 180 185 190 Tyr LeuLeu Pro Val Ala Val Val Ser Leu Ala Tyr Gly Arg Thr Leu 195 200 205 CysLeu Leu Trp Ala Ala Val Gly Pro Ala Gly Ala Ala Ala Ala Glu 210 215 220Ala Arg Arg Arg Ala Thr Gly Arg Ala Gly Arg Ala Met Leu Ala Val 225 230235 240 Ala Ala Leu Tyr Ala Leu Cys Trp Gly Pro His His Ala Leu Ile Leu245 250 255 Cys Phe Trp Tyr Gly Arg Phe Ala Phe Ser Pro Ala Thr Tyr AlaIle 260 265 270 Arg Leu Ala Ser His Cys Leu Ala Tyr Ala Asn Ser Cys LeuAsn Pro 275 280 285 Leu Val Tyr Ser Leu Ala Ser Arg His Phe Arg Ala ArgPhe Arg Arg 290 295 300 Leu Trp Pro Cys Gly Arg Cys Arg His Arg His HisHis Arg Ala His 305 310 315 320 Arg Ala Leu Arg Arg Val Gln Pro Ala SerSer Gly Pro Ala Gly Tyr 325 330 335 Pro Gly Asp Ala Arg Pro Arg Gly TrpSer Met Glu Pro Arg Gly Asp 340 345 350 Ala Leu Arg Gly Gly Gly Glu ThrArg Leu Thr Leu Ser Pro Arg Gly 355 360 365 Pro Gln 370 1275 base pairsnucleic acid double linear 3 ATG ACT TTG GCT CTG CTC TCC CCT CCT CCA TCTCCC ACG AGC TTC CAG 48 Met Thr Leu Ala Leu Leu Ser Pro Pro Pro Ser ProThr Ser Phe Gln 1 5 10 15 CCC AGA ACA CCT GGC CAG ACC CAG GTC GGG GGAGTT AGA TCC CGG GGT 96 Pro Arg Thr Pro Gly Gln Thr Gln Val Gly Gly ValArg Ser Arg Gly 20 25 30 CAA GCA ACC AGA ACT GGG GGC TCT TGC CTG AGG ATTCCA GCT TCT CTT 144 Gln Ala Thr Arg Thr Gly Gly Ser Cys Leu Arg Ile ProAla Ser Leu 35 40 45 CCC AGG TGC CCG TCT GAT GGG GAG ATG GCT GAT GCC CAGAAC ATT TCA 192 Pro Arg Cys Pro Ser Asp Gly Glu Met Ala Asp Ala Gln AsnIle Ser 50 55 60 CTG GAC AGC CCA GGG AGT GTG GGG GCC GTG GCA GTG CAT GTGGTC TTT 240 Leu Asp Ser Pro Gly Ser Val Gly Ala Val Ala Val His Val ValPhe 65 70 75 80 GCC CTA ATC TTC CTG CTG GGC ACA GTG GGC AAT GGG CTG GTGCTG GCA 288 Ala Leu Ile Phe Leu Leu Gly Thr Val Gly Asn Gly Leu Val LeuAla 85 90 95 GTG CTC CTG CAG CCT GGC CCG AGT GCC TGG CAG GAG CCT TGC AGCACC 336 Val Leu Leu Gln Pro Gly Pro Ser Ala Trp Gln Glu Pro Cys Ser Thr100 105 110 ACG GAC CTG TTC ATC CTC AAC CTG GCG GTG GCT GAC CTC TGC TTCATC 384 Thr Asp Leu Phe Ile Leu Asn Leu Ala Val Ala Asp Leu Cys Phe Ile115 120 125 CTG TGC TGC GTG CCC TTC CAA GCC ACC ATC TAC ACG CTG GAT GCCTGG 432 Leu Cys Cys Val Pro Phe Gln Ala Thr Ile Tyr Thr Leu Asp Ala Trp130 135 140 CTC TTT GGG GCC CTC GTC TGC AAC GCC GTG CAC CTG CTC ATC TACCTC 480 Leu Phe Gly Ala Leu Val Cys Asn Ala Val His Leu Leu Ile Tyr Leu145 150 155 160 ACC ATG TAC GCC AGC AGC TTT ACG CTG GCT GCT GTC TCC GTGGAC AGG 528 Thr Met Tyr Ala Ser Ser Phe Thr Leu Ala Ala Val Ser Val AspArg 165 170 175 TAC CTG GCC GTG CGG CAC CCG CTG CGC TCG CGC GCC CTG CGCACG CCG 576 Tyr Leu Ala Val Arg His Pro Leu Arg Ser Arg Ala Leu Arg ThrPro 180 185 190 CGT AAC GCC CGC GCC GCA GTG GGG CTG GTG TGG CTG CTG GCGGCG CTC 624 Arg Asn Ala Arg Ala Ala Val Gly Leu Val Trp Leu Leu Ala AlaLeu 195 200 205 TTC TCG GCG CCC TAC CTC AGC TAC TAC GGC ACC GTG CGC TACGGC GCG 672 Phe Ser Ala Pro Tyr Leu Ser Tyr Tyr Gly Thr Val Arg Tyr GlyAla 210 215 220 CTG GAG CTC TGC GTG CCC GCC TGG GAG GAC GCG CGC CGC CGCGCC CGG 720 Leu Glu Leu Cys Val Pro Ala Trp Glu Asp Ala Arg Arg Arg AlaArg 225 230 235 240 GAC GTG GCC ACC TAC GCT GCC GGC TAC CTG CTG CCC GTGGCC GTG GTG 768 Asp Val Ala Thr Tyr Ala Ala Gly Tyr Leu Leu Pro Val AlaVal Val 245 250 255 AGC CTG GCC TAC GGG CGC ACG CTG CGC TTC CTG TGG GCCGCC GTG GGT 816 Ser Leu Ala Tyr Gly Arg Thr Leu Arg Phe Leu Trp Ala AlaVal Gly 260 265 270 CCC GCG GGC GCG GCG GCG GCC GAG GCG CGG CGG AGG GCGACG GGC CGC 864 Pro Ala Gly Ala Ala Ala Ala Glu Ala Arg Arg Arg Ala ThrGly Arg 275 280 285 GCG GGG CGC GCC ATG CTG GCG GTG GCC GCG CTC TAC GCGCTC TGC TGG 912 Ala Gly Arg Ala Met Leu Ala Val Ala Ala Leu Tyr Ala LeuCys Trp 290 295 300 GGT CCG CAC CAC GCG CTC ATC CTG TGC TTC TGG TAC GGCCGC TTC GCC 960 Gly Pro His His Ala Leu Ile Leu Cys Phe Trp Tyr Gly ArgPhe Ala 305 310 315 320 TTC AGC CCG GCC ACC TAC GCC TGC CGC CTG GCC TCACAC TGC CTG GCC 1008 Phe Ser Pro Ala Thr Tyr Ala Cys Arg Leu Ala Ser HisCys Leu Ala 325 330 335 TAC GCC AAC TCC TGC CTC AAC CCG CTC GTC TAC GCGCTC GCC TCG CGC 1056 Tyr Ala Asn Ser Cys Leu Asn Pro Leu Val Tyr Ala LeuAla Ser Arg 340 345 350 CAC TTC CGC GCG CGC TTC CGC CGC CTG TGG CCG TGCGGC CGC CGA CGC 1104 His Phe Arg Ala Arg Phe Arg Arg Leu Trp Pro Cys GlyArg Arg Arg 355 360 365 CGC CAC CGT GCC CGC CGC GCT TTG CGT CGC GTC CGCCCC GCG TCC TCG 1152 Arg His Arg Ala Arg Arg Ala Leu Arg Arg Val Arg ProAla Ser Ser 370 375 380 GGC CCA CCC GGC TGC CCC GGA GAC GCC CGG CCT AGCGGG GGG CTG CTG 1200 Gly Pro Pro Gly Cys Pro Gly Asp Ala Arg Pro Ser GlyGly Leu Leu 385 390 395 400 GCT GGT GGC GGC CAG GGC CCG GAG CCC AGG GAGGGA CCC GTC CAC GGC 1248 Ala Gly Gly Gly Gln Gly Pro Glu Pro Arg Glu GlyPro Val His Gly 405 410 415 GGA GAG GCT GCC CGA GGA CCG GAA TAA 1275 GlyGlu Ala Ala Arg Gly Pro Glu 420 424 amino acids amino acid linear 4 MetThr Leu Ala Leu Leu Ser Pro Pro Pro Ser Pro Thr Ser Phe Gln 1 5 10 15Pro Arg Thr Pro Gly Gln Thr Gln Val Gly Gly Val Arg Ser Arg Gly 20 25 30Gln Ala Thr Arg Thr Gly Gly Ser Cys Leu Arg Ile Pro Ala Ser Leu 35 40 45Pro Arg Cys Pro Ser Asp Gly Glu Met Ala Asp Ala Gln Asn Ile Ser 50 55 60Leu Asp Ser Pro Gly Ser Val Gly Ala Val Ala Val His Val Val Phe 65 70 7580 Ala Leu Ile Phe Leu Leu Gly Thr Val Gly Asn Gly Leu Val Leu Ala 85 9095 Val Leu Leu Gln Pro Gly Pro Ser Ala Trp Gln Glu Pro Cys Ser Thr 100105 110 Thr Asp Leu Phe Ile Leu Asn Leu Ala Val Ala Asp Leu Cys Phe Ile115 120 125 Leu Cys Cys Val Pro Phe Gln Ala Thr Ile Tyr Thr Leu Asp AlaTrp 130 135 140 Leu Phe Gly Ala Leu Val Cys Asn Ala Val His Leu Leu IleTyr Leu 145 150 155 160 Thr Met Tyr Ala Ser Ser Phe Thr Leu Ala Ala ValSer Val Asp Arg 165 170 175 Tyr Leu Ala Val Arg His Pro Leu Arg Ser ArgAla Leu Arg Thr Pro 180 185 190 Arg Asn Ala Arg Ala Ala Val Gly Leu ValTrp Leu Leu Ala Ala Leu 195 200 205 Phe Ser Ala Pro Tyr Leu Ser Tyr TyrGly Thr Val Arg Tyr Gly Ala 210 215 220 Leu Glu Leu Cys Val Pro Ala TrpGlu Asp Ala Arg Arg Arg Ala Arg 225 230 235 240 Asp Val Ala Thr Tyr AlaAla Gly Tyr Leu Leu Pro Val Ala Val Val 245 250 255 Ser Leu Ala Tyr GlyArg Thr Leu Arg Phe Leu Trp Ala Ala Val Gly 260 265 270 Pro Ala Gly AlaAla Ala Ala Glu Ala Arg Arg Arg Ala Thr Gly Arg 275 280 285 Ala Gly ArgAla Met Leu Ala Val Ala Ala Leu Tyr Ala Leu Cys Trp 290 295 300 Gly ProHis His Ala Leu Ile Leu Cys Phe Trp Tyr Gly Arg Phe Ala 305 310 315 320Phe Ser Pro Ala Thr Tyr Ala Cys Arg Leu Ala Ser His Cys Leu Ala 325 330335 Tyr Ala Asn Ser Cys Leu Asn Pro Leu Val Tyr Ala Leu Ala Ser Arg 340345 350 His Phe Arg Ala Arg Phe Arg Arg Leu Trp Pro Cys Gly Arg Arg Arg355 360 365 Arg His Arg Ala Arg Arg Ala Leu Arg Arg Val Arg Pro Ala SerSer 370 375 380 Gly Pro Pro Gly Cys Pro Gly Asp Ala Arg Pro Ser Gly GlyLeu Leu 385 390 395 400 Ala Gly Gly Gly Gln Gly Pro Glu Pro Arg Glu GlyPro Val His Gly 405 410 415 Gly Glu Ala Ala Arg Gly Pro Glu 420 25 basepairs nucleic acid single linear 5 GCTGGCAGTG CTCCTGCAGC CTGGC 25 27base pairs nucleic acid single linear 6 AAGCGGCCGT ACCAGAAGCA CAGGATG 2726 base pairs nucleic acid single linear 7 TGCGGGCCCC AGCAGAGCGC GTAGAG26 27 base pairs nucleic acid single linear 8 CCATCCTAAT ACGACTCACTATAGGGC 27 24 base pairs nucleic acid single linear 9 CATCCAGTGTGTAGATGGCT GCCT 24 23 base pairs nucleic acid single linear 10ACTCACTATA GGGCTCGAGC GGC 23 26 base pairs nucleic acid single linear 11CCAAGTGCCT GGCAGGAGCC AAGCAG 26 28 base pairs nucleic acid single linear12 CGCGTAGAGC GCGGCCACTG CCAGCATG 28 26 base pairs nucleic acid singlelinear 13 CAAGGGCTGA ATCAANAAGC TCCAGC 26 28 base pairs nucleic acidsingle linear 14 GCGGGTTAAG GCANGAGTTG GCGTAGGC 28 25 base pairs nucleicacid single linear 15 GTGGTCTTTG CCCTAATCTT CCTGC 25 25 base pairsnucleic acid single linear 16 ACGGAGACAG CAGCCAGCGT AAAGC 25 25 basepairs nucleic acid single linear 17 CTTGGAAGGG CACGCAGCAC AGGAT 25 25base pairs nucleic acid single linear 18 GCCGCAGAGG TCAGCCACCG CCAGG 2525 base pairs nucleic acid single linear 19 TGGGCACAGT GGGCAATGGG CTGGT25 25 base pairs nucleic acid single linear 20 GAAACTGAGG AACTCTCACCCCTTG 25 25 base pairs nucleic acid single linear 21 TTGCAGGCGGCAGGGTTTAT TCCGG 25 26 base pairs nucleic acid single linear 22CAAGCAACCA GAACTGGGGG CTCTTG 26

What is claimed is:
 1. An isolated mammalian galanin receptor comprising an amino acid sequence defined by SEQ ID NO: 2 or SEQ ID NO: 4, or a conservative or allelic variant thereof.
 2. An antibody which specifically binds to the mammalian receptor of claim
 1. 3. The antibody of claim 2 which is a monoclonal antibody.
 4. An anti-idiotypic antibody produced against the antibody of claim
 2. 5. The antibody of claim 4 which is a monoclonal antibody.
 6. An isolated or recombinant nucleic acid encoding the mammalian galanin receptor of claim
 1. 7. A recombinant vector comprising the nucleic acid of claim
 6. 8. A host cell comprising the recombinant vector of claim
 7. 9. A method for making a mammalian galanin receptor comprising culturing a host cell of claim 8 under conditions in which the nucleic acid is expressed.
 10. The method of claim 9 in which the receptor is isolated from the culture.
 11. An isolated or recombinant nucleic acid selected from the group consisting of: (a) a nucleic acid encoding a mammalian galanin receptor comprising an amino acid sequence defined by SEQ ID NO: 2 or SEQ ID NO: 4, or a subsequence thereof; (b) a nucleic acid that hybridizes under moderately stringent conditions to the nucleic acid of (a) and encodes a polypeptide that (i) binds galanin and (ii) is at least 80% identical to a receptor encoded by the nucleic acid of (a); and (c) a nucleic acid that, due to the degeneracy of the genetic code, encodes a mammalian galanin receptor encoded by a nucleic acid of (a) or (b).
 12. A recombinant vector comprising the nucleic acid of claim
 11. 13. A host cell comprising the recombinant vector of claim
 12. 14. A method for making a mammalian galanin receptor comprising culturing a host cell of claim 13 under conditions in which the nucleic acid is expressed.
 15. The method of claim 7 in which the receptor is isolated from the culture.
 16. A method for identifying an agonist or antagonist of a mammalian galanin receptor, comprising: (a) contacting a mammalian galanin receptor having an amino acid sequence defined by SEQ ID NO: 2 or SEQ ID NO: 4, or a subsequence thereof, in the presence of a known amount of labled galanin with a sample to be tested for the presence of a galanin agonist or antagonist; and (b) measuring the amount of labeled galanin specifically bound to the receptor; whereby a galanin agonist or antagonist in the sample is identified by measuring substantially reduced binding of the labeled galanin to the receptor, compared to what would be measured in the absence of such agonist or antagonist.
 17. The method of claim 16 in which membranes isolated from mammalian cells comprising a nucleic acid encoding the galanin receptor are used as the source of said receptor.
 18. The method of claim 16 which further comprises: (c) contacting a mammalian GalR1 or GalR2 receptor in the presence of a known amount of labled galanin with a compound identified as a galanin agonist or antagonist in steps (a) and (b); and (d) measuring the amount of labeled galanin bound to the receptor; whereby a galanin agonist or antagonist specific for a mammalian GalR3 receptor is identified by measuring substantially undiminished binding of the labeled galanin to the receptor; compared to what would be measured in the absence of such agonist or antagonist.
 19. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an antibody or antigen-binding fragment thereof which specifically binds to a mammalian galanin receptor comprising an amino acid sequence defined by SEQ ID NO: 4 and blocks the binding of galanin thereto.
 20. The pharmaceutical composition of claim 19 which comprises a monoclonal antibody or an antigen-binding fragment thereof. 